Peptide carrier fusion proteins as allergy vaccines

ABSTRACT

The present invention relates to a polypeptide comprising at least three peptide fragments consisting of 10 to 50 consecutive amino acid residues of at least one wild-type allergen fused to the N- and C-terminus of a surface polypeptide of a virus of the hepadnaviridae family or at least one fragment of said surface polypeptide.

The present invention relates to novel polypeptides and uses thereof.

Type I allergy is an IgE-mediated hypersensitivity disease affectingalmost 25% of the population. It is based on the recognition of harmlessairborne, insect, venom, food allergen and contact allergen antigensderived from per se harmless antigen sources such as pollen, insects,mold and animal proteins by specific immunoglobulin E. The crosslinkingof effector cell-bound IgE antibodies leads to a release of inflammatorymediators (e.g., histamine, leucotrienes) and thus to the immediatesymptoms of allergy (e.g., rhinoconjunctivitis, asthma, dermatitis,anaphylaxis). T-cell activation via IgE-dependent as well asIgE-independent mechanisms contributes to chronic allergic inflammation.

The probably only causative form of allergy treatment isallergen-specific immunotherapy, which is based on the repeatedadministration of increasing amounts of allergen extracts for mostsources. Numerous clinical studies have documented the clinical efficacyof injection immunotherapy and there is evidence for severalimmunological mechanisms underlying this treatment. Due to thedifficulty to prepare high quality allergen extracts for certainallergen sources and the fact that the administration of allergens topatients can cause severe side effects, allergen-specific immunotherapycan only be recommended for certain patients groups and diseasemanifestations. It is especially difficult to treat patients withco-sensitizations to several different allergen sources and patientssuffering from severe disease manifestations such as allergic asthma.Allergic asthma is one of the most vigorous manifestations of allergy,because it severely affects the quality of daily life, causes a highrate of hospitality admissions and can manifest itself in serious,life-threatening forms requiring intensive care of the patient.

Allergen extracts prepared from natural allergen-sources are crude innature, and it is impossible to influence the quality and amounts ofindividual allergens in such preparations by technical means. They alsocontain numerous undefined non-allergenic components, and several recentstudies indicate the poor quality of such extracts and document theirgreat heterogeneity.

In the last decade great progress has been made in the field ofmolecular allergen characterization using recombinant DNA technology. Alarge number of the most important disease-eliciting allergens has beencharacterized down to the molecular level, and recombinant allergensmimicking the epitope complexity of natural allergen extracts have beenproduced. Moreover, several research groups have used the knowledgeregarding allergen structures to develop defined new allergy vaccines.Genetic engineering, synthetic peptide chemistry and conjugation ofallergens with immunostimulatory DNA sequences have been used to reducethe allergenic activity of the new vaccines and thus the rate oftherapy-induced side effects. First promising clinical studies wereconducted with such allergen derivatives. Interestingly, it turned outthat although IgE-reactivity of genetically engineered recombinantallergens and allergen-derived synthetic T-cell epitope-containingpeptides could be strongly reduced or even abolished, these derivativesstill could induce systemic side effects appearing several hours afterinjection. For example, it was reported that T-cell epitope peptides ofthe major cat allergen, Fel d 1, induced asthma and bronchial hyperreactivity several hours after intracutaneous injection, and there isstrong evidence that this effect is T-cell mediated and MHC-restricted.

These results indicate that the removal of IgE-reactivity diminishesIgE-mediated side effects since no immediate reactions were recorded inthe course of these immunotherapy studies. However, theallergen-specific T-cell epitopes which have been preserved in therecombinant allergen derivatives as well as in the peptide mixtures areresponsible for the late side effects (e.g. very problematic or atopicdermatitis, chronic T-cell-mediated allergic skin manifestation). Theside effects caused in the case of recombinant allergen-derivatives wererelatively mild and in the case of the T-cell peptide vaccines may beovercome by adequate dosing. Both of the two new approaches thereforeseem very promising for immunotherapy of allergic rhinoconjunctivitisbut may have limitations when it comes to the treatment of severe formsof allergic asthma, where the induction of late side effects in the lungmay be very problematic.

In order to administer and consequently to provoke an efficient immuneresponse against peptides, polypeptides and proteins, adjuvants and/orcarriers are regularly used. Complete Freund's adjuvant (CFA), forinstance, is one of the most potent adjuvants available. There exists aneed for vaccine compositions able to induce strong immune responsesagainst peptides and polypeptides derived from allergens and of courseof other antigens with or without the use of complete Freund's adjuvant.Further, while BSA has been used successfully as a carrier in animalmodels it may not be appropriate for use in human vaccine compositionsbecause of the risk of adverse reactions such as the risk oftransmitting prion disease (variant Creutzfeldt-Jakob disease). Afurther challenge to the development of an effective vaccine againstallergens is the need for an immune response able to rapidly decreaseallergens in an individual or animal. Therefore, high concentrations ofallergen-specific antibodies in the blood, which are mainly of the IgGsubtype, are needed. In mucosal surfaces IgA antibodies are alsoimportant.

Cholera toxin, a known carrier protein in the art, is also usedregularly as an adjuvant. However, cholera toxin increases total andspecific IgE antibody levels and leads to IgE-associated inflammatoryreactions.

Due to the side effects provoked by most carrier proteins used forvaccination, there exists a need for carrier systems which are able tostimulate immune responses against allergens or other antigens, withoutusing toxic adjuvants, without using poorly tolerated carrier proteinsand, in certain situations, without stimulation of potentiallypathologic immune responses. Novel carrier systems meeting thesespecifications can be used towards the formation of novel conjugates andcompositions suitable for the treatment or prevention of diseases likeallergic diseases.

In Bohle B. et al. (J. Immunol. 172 (11) (2004): 6642-6648) arecombinant fusion protein comprising an S-layer protein moiety and Betv 1 moiety is described. This molecule comprises the native Bet v 1allergen including Bet v 1-specific T cell epitopes.

WO 2004/004761 relates to virus like particles which are fused to animmunogen and which may be used for immunisation.

In WO 2004/003143 the use of fusion proteins comprising a virus likeparticle and an allergenic molecule as immunogen for vaccination isdisclosed.

In WO 2007/140505 and Edlmayr et al. (J. Immunol. 182 (10) (2009)6298-6306) the use of fusion proteins comprising vartious carriermolecules fused to allergen-derived peptides are described to induceallergen-specific IgG antibodies but these constructs do not exhibit animmunomodulatory effect which may be considered advantageous forallergic patients such as the induction of IL-10 or Th1 immunity. FIG. 4of Edlmayr et al shows that KLH-fused peptides induce the Th2 cytokineIL-5 and VP1 fusion proteins do not induce IL-10 or IFN-gamma.

In Niespodziana et al (J. Allergy Clin. Immunol. 127 (6) (2011)1562-1570) the use of fusion proteins each comprising HepatitisB-derived PreS and two peptides derived from the major cat allergen Feld 1 are described to induce allergen-specific IgG antibodies. However,no regimen suitable for vaccination of humans has been described and thepeptides contained allergen-specific T cell epitopes.

It is an object of the present invention to provide medicaments andcarriers which overcome the aforementioned drawbacks and allow anallergen vaccination with reduced side effects.

Therefore, the present invention relates to a polypeptide comprising atleast three peptide fragments consisting of 10 to 50 consecutive aminoacid residues of at least one wild-type allergen fused to the N- andC-terminus of a surface polypeptide of a virus of the hepadnaviridaefamily or at least one fragment of said surface polypeptide orcomprising a surface polypeptide of a virus of the hepadnaviridae familyor at least one fragment thereof fused N- and/or C-terminally to atleast three peptides derived from at least one wild-type allergen.

In order to provoke an enhanced immune response against a molecule, inparticular against an allergenic or hypoallergenic molecule according tothe present invention, at least three peptide fragments derived from atleast one wild-type allergen are fused (by genetic engineering) to asurface polypeptide of a virus of the hepadnaviridae family, preferablyof a Hepatitis B virus, more preferably of a Hepatitis B PreSpolypeptide, or at least one fragment thereof. It turned surprisinglyout that in contrast to conventionally and regularly employed carrierproteins like KLH (Keyhole limpet hemocyanin) a surface polypeptide of avirus of the hepadnaviridae family, preferably of a Hepatitis B virus,more preferably of a Hepatitis B PreS polypeptide, or fragments thereoflead to an enhanced formation of antibodies directed to those peptideswhich are bound thereto.

Moreover, it turned out that allergen specific IgG antibodies induced byimmunization with more than three properly selected allergen derivedpeptide fragmentsfused to the Hepatitis B PreS polypeptide are betterfocused to the IgE epitopes of the allergen while immunization with thewild-type allergen leads to IgG which are directed to all parts of theallergen—also those which are not IgE reactive. In an experimentnormalized for IgG titers this leads to a better blocking capacity ofPreS/peptide induced IgG compared to wild-type allergen induced (FIG.12).

Also very surprisingly, it turned out that in cultures of human PBMCsfusion proteins of allergen derived peptide fragments to the Hepatitis BPreS polypeptide strongly induced the cytokines IL-10 and IFN-gamma,which have been attributed as positive indicators for a successfulallergy immunotherapy. In contrast, induction of IL-10 and IFN-gamma wassignificantly lower with wild-type allergen, allergen derived peptidefragments alone or PreS alone (FIGS. 10 A-10 C).

“Fused to the N- and C-terminus”, as used herein, means that at leastone peptide is fused to the N-terminus of a surface polypeptide of avirus of the hepadnaviridae family or at least one fragment of saidsurface polypeptide and at least one peptide is fused to the C-terminusof a surface polypeptide of a virus of the hepadnaviridae family or atleast one fragment of said surface polypeptide. In a most simpliestembodiment of the present invention a surface polypeptide of a virus ofthe hepadnaviridae family or at least one fragment of said surfacepolypeptide may comprise at the N-terminus one peptide and on theC-terminus two peptides or vice versa.

The polypeptide of the present invention preferably comprises at leastfour, more preferably at least five, even more preferably at least six,peptide fragments, preferably B cell binding peptides, derived from anallergen, whereby four peptides are most preferred.

According to a particularly preferred embodiment of the presentinvention the carrier protein is the Hepatitis B PreS polypeptide withthe following amino acid sequence (SEQ ID No. 21):

GGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFGPGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN

It is also possible to use fragments Hepatitis B PreS1 or Hepatitis BPreS2 of the Hepatitis B PreS polypeptide. A fragment of the Hepatitis BPreS polypeptide preferably comprises or consists of at least 30,preferably at least 40, more preferably at least 50, consecutive aminoacid residues of SEQ ID No. 21.

“Hypoallergenic” as used herein, refers to molecules with reduced or noallergenic potential (i.e. IgE reactivity determined with IgE bindingassays known in the art). Such molecules have a decreased capacity toprovoke allergic reactions in an individual compared to the wild-typeprotein from which these molecules are derived.

The at least three, preferably at least four, more preferably at leastfive, even more preferably at least six, peptide fragments fused to theN- and C-terminus of a surface polypeptide of a virus of thehepadnaviridae family or at least one fragment of said surfacepolypeptide comprise or consist of 10 to 50 consecutive amino acids,more preferably 15 to 50 consecutive amino acids, in particular 20-50consecutive amino acids, of at least one wild-type allergen and exhibitpreferably reduced IgE reactivity compared to the wild-type allergenfrom which the peptide fragments are derived from. These peptidefragments are preferably designed to exclude allergen-specific T-cellepitopes which may cause T-cell-mediated side effects. T-cell epitopesand molecules exhibiting reduced T-cell response may be determined andidentified by methods known by the person skilled in the art (e.g.,Bercovici N. et al. Clin Diagn Lab Immunol. (2000) 7:859-864).

The at least three peptide fragments comprising or consisting of 10 to50 consecutive amino acids, more preferably 15 to 50 consecutive aminoacids, in particular 20-50 consecutive amino acids, of at least onewild-type allergen can be derived from one and the same allergen. If twoor more fragments are derived from the same allergen these two or morefragments are not adjacently located in the wild type allergen and/orhave an order in the polypeptide of the present invention which does notcorrespond to the order in the wild type allergen.

The term “peptide fragment” as used herein means a part/fragment of ahypoallergenic polypeptide or fusion protein of the invention which isderived from the primary structure of a wild-type allergen and compriseor consist of 10 to 50 consecutive amino acids, more preferably 15 to 50consecutive amino acids, in particular 20-50 consecutive amino acids, ofthis wild-type allergen.

The terms “derived from an allergen” and “derived from at least onewild-type allergen”, as used herein, mean that the peptide fragmentsaccording to the present invention are obtained directly from anallergen by fragmentation or truncation. The amino acid sequence ofthese peptide fragments is preferably at least 80% identical, morepreferably at least 90% identical, most preferably at least 95%identical, in particular 100% identical, to the amino sequence stretchof the wild-type allergen, from which the peptide fragments are derivedfrom. However, the peptides which are not 100% identical to thewild-type allergen fragments should be able to bind with at least 60%,preferably at least 70%, more preferably at least 80%, most preferablyat least 90%, strength to an antibody or to antibodies, preferably toIgG antibodies, which are directed to said wild-type allergen fragments.“At least one wild-type allergen” means that the polypeptide of thepresent invention may comprise B-cell binding peptides of more than one,preferably two, more preferably three, different wild-type allergens(i.e. sources) (e.g. one peptide is derived from Bet v 1, one from Amb a1 and one from Phl p 1 or two peptides are derived from Bet v 1 and onefrom Amb a 1).

The degree of identity of a first amino acid sequence to a second aminoacid can be determined by a direct comparison between both amino acidsequences using certain algorithms. Such algorithms are, for instance,incorporated in various computer programs (e.g. “BLAST 2 SEQUENCES(blastp)” (Tatusova et al. (1999) FEMS Microbiol. Lett. 174:247-25;Corpet F, Nucl. Acids Res. (1988) 16:10881-10890).

The polypeptides of the present invention may be obtained by recombinantmethods or chemical synthesis. Alternatively, it is, of course, alsopossible to obtain the molecules by enzymatic or chemical cleavage ofthe wild-type allergen or a polypeptide/protein harbouring the moleculeof interest.

It was now surprisingly found that peptide carrier fusion proteins withimproved properties can be obtained by employing surface proteins fromviruses of the hepadnaviridae class, more specifically the humanhepatitis B virus. One up to 20, preferably 3 or 4 up to 20, morepreferably 3 or 4 up to 15, even more preferably 3 or 4 up to 10 (i.e.3, 4, 5, 6, 7, 8, 9, 10), peptide fragments, preferably hypoallergenicpeptide fragments, can be fused to the C-terminus and the N-terminus ofa surface polypeptide of a virus of the hepadnaviridae family or atleast one fragment of said surface polypeptide. A preferred embodimentof the current invention are therefore fusion proteins composed of atleast 3 up to 6 hypoallergenic peptide fragments with a carrier proteinderived from the surface antigens of human hepatitis B virus. Accordingto a particularly preferred embodiment of the present invention suchfusion proteins use the preS protein as carrier. A most preferredembodiment of this invention are fusion proteins where 4 hypoallergenicpeptide fragments are fused to the preS carrier protein or a fragmentthereof. The (hypoallergenic) peptide fragments can be the same ordifferent and can derived from one or several allergenic proteins andthe locus of the peptides within the fusion protein is the C-terminusand the N-terminus of the carrier protein. One up to three(hypoallergenic) peptide fragments can be fused to each of theC-terminus and the N-terminus in such a way that the sum of the(hypoallergenic) peptide fragments will be, for instance, three or fourto six. The terms “fused” or “fusion protein”, refer to a preferredembodiment of the invention, meaning that the non-allergenic carrierprotein and the (hypoallergenic) peptide fragments at the carrier's C-and N-terminus are expressed and prepared as one singular recombinantpolypeptide chain

A most highly preferred embodiment of the current invention are fusionproteins of the hepatitis B virus preS protein, which carry(hypoallergenic) peptide fragments derived from a specific allergen,such that one or two, preferably two, peptide fragments each are fusedto the C-terminus and the N-terminus of the carrier. For illustration,the preferred polypeptides of the current invention may have the generalmolecular structure represented by the following generic structures:

It is understood that peptides A, B, C and D can be the same ordifferent and may be derived from the same allergen for each individualfusion protein or will be derived from different allergens.

The (hypoallergenic) peptides to be fused to the N- and C-terminus ofthe surface polypeptide of a virus of the hepadnaviridae family or atleast one fragment of said surface polypeptide, preferably the preSprotein or a fragment thereof, are preferably selected from the groupconsisting of major birch pollen allergens, in particular Bet v 1 andBet v 4, major timothy grass pollen allergens, in particular Phl p 1,Phl p 2, Phl p 5, Phl p 6 and Phl p 7, major house dust mite allergens,in particular Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23,major cat allergen Fel d 1, the major ragweed allergen Amb a 1, themajor Japanese cedar allergens Cry j 1 and Cry j 2, major bee allergens,major wasp allergens, profilins, especially Phl p 7, Phl p 12.

Other suited allergens to be used according to the present invention canbe derived from the following table 2 without being restricted to saidtable.

TABLE 2 Sources of hypoallergenic peptides Allergen Biochem. ID or cDNA(C) or Reference, Species Name Name Obsolete name MW protein (P) Acc.No. Ambrosia artemisiifolia Amb a 1 antigen E  8 C 8, 20 short ragweedAmb a 2 antigen K 38 C 8, 21 Amb a 3 Ra3 11 C 22 Amb a 5 Ra5  5 C 11, 23Amb a 6 Ra6 10 C 24, 25 Amb a 7 Ra7 12 P 26 Ambrosia trifida Amb t 5Ra5G   4.4 C 9, 10, 27 giant ragweed Artemisia vulgaris Art v 1 27-29 C28 mugwort Art v 2 35 P 28A Art v 3 lipid transfer protein 12 P 53 Art v4 profilin 14 C 29 Helianthus annuus Hel a 1 34 29A sunflower Hel a 2profilin   15.7 C Y15210 Mercurialis annua Mer a 1 profilin 14-15 CY13271 Caryophyllales Che a 1 17 C 29B, AY049012 Chenopodium album Che a2 profilin 14 C AY082337 lamb's-quarters, pigweed, Che a 3 polcalcin 10C AY082338 white goosefoot Salsola kali Sal k 1 43 P 29C Russian-thistleRosales Hum j 4w C AY335187 Humulus japonicus Japanese hop Parietariajudaica Par j 1 lipid transfer protein 1 15 C see list of isoallergensPar j 2 lipid transfer protein 2 C see list of isoallergens Par j 3profilin C see list of isoallergens Parietaria officinalis Par o 1 lipidtransfer protein 15 29D B. Grasses Cyn d 1 32 C 30, S83343 Poales Cyn d7 C 31, X91256 Cynodon dactylon Cyn d 12 profilin 14 C 31a, Y08390Bermuda grass Cyn d 15  9 C AF517686 Cyn d 22w enolase data pending Cynd 23 Cyn d 14  9 C AF517685 Cyn d 24 Pathogenesis- related p. 21 Ppending Dactylis glomerata Dac g 1 AgDg1 32 P 32 orchard grass Dac g 211 C 33, S45354 Dac g 3 C 33A, U25343 Dac g 5 31 P 34 Festuca pratensisFes p 4w 60 — meadow fescue Holcus lanatus Hol l 1 C Z27084 velvet grassLolium perenne Lol p 1 group I 27 C 35, 36 rye grass Lol p 2 group II 11P 37, 37A, X73363 Lol p 3 group III 11 P 38 Lol p 5 Lol p IX, Lol p Ib31/35 C 34, 39 Lol p 11 hom: trypsin inhibitor 16 39A Phalaris aquaticaPha a 1 C 40, S80654 canary grass Phleum pratense Phl p 1 27 C X78813timothy Phl p 2 C X75925, 41 Phl p 4 P 41A Phl p 5 Ag25 32 C 42 Phl p 6C Z27082, 43 Phl p 11 trypsin inhibitor hom. 20 C AF521563, 43A Phl p 12profilin C X77583, 44 Phl p 13 polygalacturonase 55-60 C AJ238848 Poapratensis Poa p 1 group I 33 P 46 Kentucky blue grass Poa p 5 31/34 C34, 47 Sorghum halepense Sor h 1 C 48 Johnson grass C. Trees Pho d 2profilin   14.3 C Asturias p.c. Arecales Phoenix dactylifera date palmFagales Aln g 1 17 C S50892 Alnus glutinosa alder Betula verrucosa Bet v1 17 C see list of isoallergens birch Bet v 2 profilin 15 C M65179 Bet v3 C X79267 Bet v 4  8 C X87153, S54819 Bet v 6 h: isoflavone reductase  33.5 C see list of isoallergens Bet v 7 cyclophilin 18 P P81531Carpinus betulus Car b 1 17 C see list of isoallergens hornbeam Castaneasativa Cas s 1 22 P 52 chestnut Cas s 5 chitinase Cas s 8 lipid transferprotein   9.7 P 53 Corylus avellana Cor a 1 17 C see list ofisoallergens hazel Cor a 2 profilin 14 C Cor a 8 lipid transfer protein9 C Cor a 9 11S globulin-like protein 40/?  C Beyer p.c. Cor a 10luminal binding prot. 70 C AJ295617 Cor a 11 7S vicilin-like prot. 48 CAF441864 Quercus alba Que a 1 17 P 54 White oak Lamiales Fra e 1 20 P58A, AF526295 Oleaceae Fraxinus excelsior ash Ligustrum vulgare Lig v 120 P 58A privet Olea europea Ole e 1 16 C 59, 60 olive Ole e 2 profilin15-18 C 60A Ole e 3   9.2 60B Ole e 4 32 P P80741 Ole e 5 superoxidedismutase 16 P P80740 Ole e 6 10 C 60C, U86342 Ole e 7 ? P 60D, P81430Ole e 8 Ca2+-binding protein 21 C 60E, AF078679 Ole e 9beta-1,3-glucanase 46 C AF249675 Ole e 10 glycosyl hydrolase hom. 11 C60F, AY082335 Syringa vulgaris Syr v 1 20 P 58A lilac Plantaginaceae Plal 1 18 P P842242 Plantago lanceolata English plantain Pinales Cry j 141-45 C 55, 56 Cryptomeria japonica Cry j 2 C 57, D29772 sugi Cupressusarisonica Cup a 1 43 C A1243570 cypress Cupressus sempervirens Cup s 143 C see list of isoallergens common cypress Cup s 3w 34 C ref pendingJuniperus ashei Jun a 1 43 P P81294 mountain cedar Jun a 2 C 57A,AJ404653 Jun a 3 30 P 57B, P81295 Juniperus oxycedrus Jun o 4 hom:calmodulin 29 C 57C, AF031471 prickly juniper Juniperus sabinoides Jun s1 50 P 58 mountain cedar Juniperus virginiana Jun v 1 43 P P81825, 58Beastern red cedar Platanaceae Pla a 1 18 P P82817 Platanus acerifoliaPla a 2 43 P P82967 London plane tree Pla a 3 lipid transfer protein 10P Iris p.c. D. Mites Aca s 13 arthropod  14* C AJ006774 Acarus sirofatty acid binding prot. mite Blomia tropicalis Blo t 1 cysteineprotease 39 C AF277840 mite Blo t 3 trypsin  24* C Cheong p.c. Blo t 4alpha amylase 56 C Cheong p.c. Blo t 5 C U59102 Blo t 6 chymotrypsin 25C Cheong p.c. Blo t 10 tropomyosin 33 C 61 Blo t 11 paramyosin 110  CAF525465, 61A Blo t 12 Bt11a C U27479 Blo t 13 Bt6, fatty acid bindprot. C U58106 Blo t 19 anti-microbial pep. hom.   7.2 C Cheong p.c.Dermatophagoides farinae Der f 1 cysteine protease 25 C 69 Americanhouse dust mite Der f 2 14 C 70, 70A, see list of isoallergens Der f 3trypsin 30 C 63 Der f 7 24-31 C SW: Q26456, 71 Der f 10 tropomyosin C 72Der f 11 paramyosin 98 C 72A Der f 14 mag3, apolipophorin C D17686 Der f15 98k chitinase 98 C AF178772 Der f 16 gelsolin/villin 53 C 71A Der f17 Ca binding EF protein 53 C 71A Der f 18w 60k chitinase 60 C Weberp.c. Dermatophagoides microceras Der m 1 cysteine protease 25 P 68 housedust mite Dermatophagoides pteronyssinus Der p 1 antigen P1, cysteineprotease 25 C 62, see list of isoallergens European house dust mite Derp 2 14 C 62A-C, see list of isoallergens Der p 3 trypsin 28/30 C 63 Derp 4 amylase 60 P 64 Der p 5 14 C 65 Der p 6 chymotrypsin 25 P 66 Der p 722/28 C 67 Der p 8 glutathione transferase C 67A Der p 9 collagenolyticserine pro. P 67B Der p 10 tropomyosin 36 C Y14906 Der p 14apolipophorin like prot. C Epton p.c. Euroglyphus maynei Eur m 2 C seelist of isoallergens mite Eur m 14 apolipophorin 177  C AF149827Glycyphagus domesticus Gly d 2 C 72B, see isoallergen list storage miteLepidoglyphus destructor Lep d 2 15 C 73, 74, 74A, see isoallergen liststorage mite Lep d 1 Lep d 5 C 75, AJ250278 Lep d 7 C 75, AJ271058 Lep d10 tropomyosin C 75A, AJ250096 Lep d 13 C 75, AJ250279 Tyrophagusputrescentiae Tyr p 2 C 75B, Y12690 storage mite E. Animals Bos d 2 Ag3,lipocalin 20 C 76, see isoallergen list Bos domesticus Bos d 3Ca-binding S100 hom. 11 C L39834 domestic cattle Bos d 4alpha-lactalbumin   14.2 C M18780 (see also foods) Bos d 5beta-lactoglobulin   18.3 C X14712 Bos d 6 serum albumin 67 C M73993 Bosd 7 immunoglobulin 160  77 Bos d 8 caseins 20-30 77 Canis familiaris Canf 1 25 C 78, 79 (Canis domesticus) Can f 2 27 C 78, 79 dog Can f 3albumin C S72946 Can f 4 18 P A59491 Equus caballus Equ c 1 lipocalin 25C U70823 domestic horse Equ c 2 lipocalin   18.5 P 79A, 79B Equ c 3Ag3 - albumin 67 C 79C, X74045 Equ c 4 17 P 79D Equ c 5 AgX 17 P GoubranBotros p.c. Felis domesticus Fel d 1 cat-1 38 C 15 cat (saliva) Fel d 2albumin C 79E, X84842 Fel d 3 cystatin 11 C 79F, AF238996 Fel d 4lipocalin 22 C AY497902 Fel d 5w immunoglobulin A 400  Adedoyin p.c. Feld 6w immunoglobulin M  800-1000 Adedoyin p.c. Fel d 7w immunoglobulin G150  Adedoyin p.c. Cavia porcellus Cav p 1 lipocalin homologue 20 P SW:P83507, 80 guinea pig Cav p 2 17 P SW: P83508 Mus musculus Mus m 1 MUP19 C 81, 81A mouse (urine) Rattus norvegius Rat n 1 17 C 82, 83 rat(urine) F. Fungi (moulds) Alt a 1 28 C U82633 1. Ascomycota Alt a 2 25 C83A, U62442 1.1 Dothideales Alt a 3 heat shock prot. 70 C U87807, U87808Alternaria alternata Alt a 4 prot. disulfideisomerase 57 C X84217 Alt a6 a cid ribosomal prot. P2 11 C X78222, U87806 Alt a 7 YCP4 protein 22 CX78225 Alt a 10 aldehyde dehydrogenase 53 C X78227, P42041 Alt a 11enolase 45 C U82437 Alt a 12 acid ribosomal prot. P1 11 C X84216Cladosporium herbarum Cla h 1 13 83B, 83C Cla h 2 23 83B, 83C Cla h 3aldehyde dehydrogenase 53 C X78228 Cla h 4 acid ribosomal prot. P2 11 CX78223 Cla h 5 YCP4 protein 22 C X78224 Cla h 6 enolase 46 C X78226 Clah 12 acid ribosomal prot. P1 11 C X85180 1.2 Eurotiales Asp fl 13alkaline serine protease 34 84 Aspergillus flavus Aspergillus fumigatusAsp f 1 18 C M83781, S39330 Asp f 2 37 C U56938 Asp f 3 peroxisomalprotein 19 C U20722 Asp f 4 30 C AJ001732 Asp f 5 metalloprotease 40 CZ30424 Asp f 6 Mn superoxide dismut.   26.5 C U53561 Asp f 7 12 CAJ223315 Asp f 8 ribosomal prot. P2 11 C AJ224333 Asp f 9 34 C AJ223327Asp f 10 aspartic protease 34 C X85092 Asp f 11 peptidyl-prolylisomerase 24 84A Asp f 12 heat shock prot. P90 90 C 85 Asp f 13 alkalineserine protease 34 84B Asp f 15 16 C AJ002026 Asp f 16 43 C g3643813 Aspf 17 C AJ224865 Asp f 18 vacuolar serine protease 34 84C Asp f 22wenolase 46 C AF284645 Asp f 23 L3 ribosomal protein 44 C 85A, AF464911Aspergillus niger Asp n 14 beta-xylosidase 105  C AF108944 Asp n 18vacuolar serine protease 34 C 84B Asp n 25 3-phytase B  66-100 C 85B,P34754 Asp n ? 85 C Z84377 Aspergillus oryzae Asp o 13 alkaline serineprotease 34 C X17561 Asp o 21 TAKA-amylase A 53 C D00434, M33218Penicillium brevicompactum Pen b 13 alkaline serine protease 33 86APenicillium chrysogenum Pen ch 13 alkaline serine protease 34 87(formerly P. notatum) Pen ch 18 vacuolar serine protease 32 87 Pen ch 20N-acetyl glucosaminidase 68 87A Penicillium citrinum Pen c 3 peroxisomalmem. prot. 18 86B Pen c 13 alkaline serine protease 33 86A Pen c 19 heatshock prot. P70 70 C U64207 Pen c 22w enolase 46 C AF254643 Pen c 24elongation factor 1 beta C AY363911 Penicillium oxalicum Pen o 18vacuolar serine protease 34 87B 1.3 Hypocreales Fus c 1 ribosomal prot.P2  11* C AY077706 Fusarium culmorum Fus c 2 thioredoxin-like prot.  13*C AY077707 1.4 Onygenales Tri r 2 C 88 Trichophyton rubrum Tri r 4serine protease C 88 Trichophyton tonsurans Tri t 1 30 P 88A Tri t 4serine protease 83 C 88 1.5 Saccharomycetales Cand a 1 40 C 89 Candidaalbicans Cand a 3 peroxisomal protein 29 C AY136739 Candida boidiniiCand b 2 20 C J04984, J04985 2. Basidiomycotina Psi c 1 2.1Hymenomycetes Psi c 2 cyclophilin 16 89A Psilocybe cubensis Coprinuscomatus Cop c 1 leucine zipper protein 11 C AJ132235 shaggy cap Cop c 2AJ242791 Cop c 3 AJ242792 Cop c 5 AJ242793 Cop c 7 AJ242794 2.2Urediniomycetes Rho m 1 enolase 47 C 89B Rhodotorula mucilaginosa Rho m2 vacuolar serine protease 31 C AY547285 2.3 Ustilaginomycetes Mala f 2MF1, peroxisomal 21 C AB011804, 90 Malassezia furfur membrane proteinMala f 3 MF2, peroxisomal 20 C AB011805, 90 membrane protein Mala f 4mitochondrial malate dehydrogenase 35 C AF084828, 90A Malasseziasympodialis Mala s 1 C X96486, 91 Mala s 5  18* C AJ011955 Mala s 6  17*C AJ011956 Mala s 7 C AJ011957, 91A Mala s 8  19* C AJ011958, 91A Mala s9  37* C AJ011959, 91A Mala s 10 heat shock prot. 70 86 C AJ428052 Malas 11 Mn superoxide dismut. 23 C AJ548421 3. Deuteromycotina Epi p 1serine protease 30 P SW: P83340, 91B 3.1 Tuberculariales Epicoccumpurpurascens (formerly E. nigrum) G. Insects Aed a 1 apyrase 68 C L12389Aedes aegyptii Aed a 2 37 C M33157 mosquito Apis mellifera Api m 1phospholipase A2 16 C 92 honey bee Api m 2 hyaluronidase 44 C 93 Api m 4melittin  3 C 94 Api m 6 7-8 P Kettner p.c. Api m 7 CUB serine protease39 C AY127579 Bombus pennsylvanicus Bom p 1 phospholipase 16 P 95 bumblebee Bom p 4 protease P 95 Blattella germanica Bla g 1 Bd90k C Germancockroach Bla g 2 aspartic protease 36 C 96 Bla g 4 calycin 21 C 97 Blag 5 glutathione transferase 22 C 98 Bla g 6 troponin C 27 C 98Periplaneta americana Per a 1 Cr-PII C American cockroach Per a 3 Cr-PI72-78 C 98A Per a 7 tropomyosin 37 C Y14854 Chironomus kiiensis Chi k 10tropomyosin   32.5* C AJ012184 midge Chironomus thummi thummi Chi t 1-9hemoglobin 16 C 99 midge Chi t 1.01 component III 16 C P02229 Chi t 1.02component IV 16 C P02230 Chi t 2.0101 component I 16 C P02221 Chi t2.0102 component IA 16 C P02221 Chi t 3 component II-beta 16 C P02222Chi t 4 component IIIA 16 C P02231 Chi t 5 component VI 16 C P02224 Chit 6.01 component VIIA 16 C P02226 Chi t 6.02 component IX 16 C P02223Chi t 7 component VIIB 16 C P02225 Chi t 8 component VIII 16 C P02227Chi t 9 component X 16 C P02228 Ctenocephalides felis felis Cte f 1 catflea Cte f 2 M1b 27 C AF231352 Cte f 3 25 C Thaumetopoea pityocampa Thap 1 15 P PIR: A59396, 99A pine processionary moth Lepisma saccharina Leps 1 tropomyosin 36 C AJ309202 silverfish Dolichovespula maculata Dol m 1phospholipase A1 35 C 100 white face hornet Dol m 2 hyaluronidase 44 C101 Dol m 5 antigen 5 23 C 102, 103 Dolichovespula arenaria Dol a 5antigen 5 23 C 104 yellow hornet Polistes annularies Pol a 1phospholipase A1 35 P 105 wasp Pol a 2 hyaluronidase 44 P 105 Pol a 5antigen 5 23 C 104 Polistes dominulus Pol d 1 Hoffman p.c. Mediterraneanpaper wasp Pol d 4 serine protease 32-34 C Hoffman p.c. Pol d 5 P81656Polistes exclamans Pol e 1 phospholipase A1 34 P 107 wasp Pol e 5antigen 5 23 C 104 Polistes fuscatus Pol f 5 antigen 5 23 C 106 waspPolistes gallicus Pol g 5 antigen 5 24 C P83377 wasp Polistes metricusPol m 5 antigen 5 23 C 106 wasp Vespa crabo Vesp c 1 phospholipase 34 P107 European hornet Vesp c 5 antigen 5 23 C 106 Vespa mandarina Vesp m 1Hoffman p.c. giant asian hornet Vesp m 5 P81657 Vespula flavopilosa Vesf 5 antigen 5 23 C 106 yellowjacket Vespula germanica Ves g 5 antigen 523 C 106 yellowjacket Vespula maculifrons Ves m 1 phospholipase A1  33.5 C 108 yellowjacket Ves m 2 hyaluronidase 44 P 109 Ves m 5 antigen5 23 C 104 Vespula pennsylvanica Ves p 5 antigen 5 23 C 106 yellowjacketVespula squamosa Ves s 5 antigen 5 23 C 106 yellowjacket Vespula viduaVes vi 5 antigen 5 23 C 106 wasp Vespula vulgaris Ves v 1 phospholipaseA1 35 C 105A yellowjacket Ves v 2 hyaluronidase 44 P 105A Ves v 5antigen 5 23 C 104 Myrmecia pilosula Myr p 1 C X70256 Australian jumperant Myr p 2 C S81785 Solenopsis geminata Sol g 2 Hoffman p.c. tropicalfire ant Sol g 4 Hoffman p.c. Solenopsis invicta Sol i 2 13 C 110, 111fire ant Sol i 3 24 C 110 Sol i 4 13 C 110 Solenopsis saevissima Sol s 2Hoffman p.c. Brazilian fire ant Triatoma protracta Tria p 1 Procalin 20C AF179004, 111A. California kissing bug H. Foods Gad c 1 allergen M 12C 112, 113 Gadus callarias cod Salmo salar Sal s 1 parvalbumin 12 CX97824 Atlantic salmon Bos domesticus Bos d 4 alpha-lactalbumin   14.2 CM18780 domestic cattle Bos d 5 beta-lactoglobulin   18.3 C X14712 (milk)Bos d 6 serum albumin 67 C M73993 see also animals Bos d 7immunoglobulin 160  77 Bos d 8 caseins 20-30 77 Cyprinus carpio Cyp c 1parvalbumin 12 C 129 (Common carp) Gallus domesticus Gal d 1 ovomucoid28 C 114, 115 chicken Gal d 2 ovalbumin 44 C 114, 115 Gal d 3 Ag22,conalbumin 78 C 114, 115 Gal d 4 lysozyme 14 C 114, 115 Gal d 5 serumalbumin 69 C X60688 Metapenaeus ensis Met e 1 tropomyosin C U08008shrimp Penaeus aztecus Pen a 1 tropomyosin 36 P 116 shrimp Penaeusindicus Pen i 1 tropomyosin 34 C 116A shrimp Penaeus monodon Pen m 1tropomyosin 38 C black tiger shrimp Pen m 2 arginine kinase 40 CAF479772, 117 Todarodes pacificus Tod p 1 tropomyosin 38 P 117A squidHelix aspersa Hel as 1 tropomyosin 36 C Y14855, 117B brown garden snailHaliotis midae Hal m 1 49 117C abalone Rana esculenta Ran e 1parvalbumin alpha   11.9* C AJ315959 edible frog Ran e 2 parvalbuminbeta   11.7* C AJ414730 Brassica juncea Bra j 1 2S albumin 14 C 118oriental mustard Brassica napus Bra n 1 2S albumin 15 P 118A, P80208rapeseed Brassica rapa Bra r 2 hom: prohevein 25 P81729 turnip Hordeumvulgare Hor v 15 BMAI-1 15 C 119 barley Hor v 16 alpha-amylase Hor v 17beta-amylase Hor v 21 gamma-3 hordein 34 C 119A, SW: P80198 Secalecereale Sec c 20 secalin see isoall. list rye Triticum aestivum Tri a 18agglutinin wheat Tri a 19 omega-5 gliadin 65 P PIR: A59156 Zea mays Zeam 14 lipid transfer prot.  9 P P19656 maise, corn Oryza sativa Ory s 1 C119B, U31771 rice Apium graveolens Api g 1 hom: Bet v 1  16* C Z48967celery Api g 4 profilin AF129423 Api g 5 55/58 P P81943 Daucus carotaDau c 1 hom: Bet v 1 16 C 117D, see isoallergen list carrot Dau c 4profilin C AF456482 Corylus avellana Cor a 1.04 hom: Bet v 1 17 C seelist of isoallergens hazelnut Cor a 2 profilin 14 C AF327622 Cor a 8lipid transfer protein  9 C AF329829 Malus domestica Mal d 1 hom: Bet v1 C see list of isoallergens apple Mal d 2 hom: thaumatin C AJ243427 Mald 3 lipid transfer protein  9 C Pastorello p.c. Mal d 4 profilin   14.4*C see list of isoallergens Pyrus communis Pyr c 1 hom: Bet v 1 18 CAF05730 pear Pyr c 4 profilin 14 C AF129424 Pyr c 5 hom: isoflavonereductas   33.5 C AF071477 Persea americana Pers a 1 endochitinase 32 CZ78202 avocado Prunus armeniaca Pru ar 1 hom: Bet v 1 C U93165 apricotPru ar 3 lipid transfer protein  9 P Prunus avium Pru av 1 hom: Bet v 1C U66076 sweet cherry Pru av 2 hom: thaumatin C U32440 Pru av 3 lipidtransfer protein 10 C AF221501 Pru av 4 profilin 15 C AF129425 Prunusdomestica Pru d 3 lipid transfer protein  9 P 119C European plum Prunuspersica Pru p 3 lipid transfer protein 10 P P81402 peach Pru p 4profilin 14 C see isoallergen list Asparagus officinalis Aspa o 1 lipidtransfer protein  9 P 119D Asparagus Crocus sativus Cro s 1 21 VarastehA-R p.c. saffron crocus Lactuca sativa Lac s 1 lipid transfer protein  9Vieths p.c. lettuce Vitis vinifera Vit v 1 lipid transfer protein  9 PP80274 grape Musa × paradisiaca Mus xp 1 profilin 15 C AF377948 bananaAnanas comosus pineapple Ana c 1 profilin 15 C AF377949 Ana c 2bromelain   22.8* C 119E-G, D14059 Citrus limon Cit l 3 lipid transferprotein  9 P Torrejon p.c. lemon Citrus sinensis Cit s 1 germin-likeprotein 23 P Torrejon p.c. sweet orange Cit s 2 profilin 14 P Torrejonp.c. Cit s 3 lipid transfer protein  9 P Torrejon p.c. Litchi chinensisLit c 1 profilin 15 C AY049013 litchi Sinapis alba Sin a 1 2S albumin 14C 120 yellow mustard Glycine max Gly m 1 HPS  7 P 120A soybean Gly m 2 8 P A57106 Gly m 3 profilin 14 C see list of isoallergens Gly m 4(SAM22) PR-10 prot. 17 C X60043, 120B Vigna radiata Vig r 1 PR-10protein 15 C AY792956 mung bean Arachis hypogaea Ara h 1 vicilin   63.5C L34402 peanut Ara h 2 conglutin 17 C L77197 Ara h 3 glycinin 60 CAF093541 Ara h 4 glycinin 37 C AF086821 Ara h 5 profilin 15 C AF059616Ara h 6 hom: conglutin 15 C AF092846 Ara h 7 hom: conglutin 15 CAF091737 Ara h 8 PR-10 protein 17 C AY328088 Lens culinaris Len c 1vicilin 47 C see list of isoallergens lentil Len c 2 seed biotinylatedprot. 66 P 120C Pisum savitum Pis s 1 vicilin 44 C see list ofisoallergens pea Pis s 2 convicilin 63 C pending Actinidia chinensis Actc 1 cysteine protease 30 P P00785 kiwi Act c 2 thaumatin-like protein 24P SW: P81370, 121 Capsicum annuum Cap a 1w osmotin-like protein 23 CAJ297410 bell pepper Cap a 2 profilin 14 C AJ417552 Lycopersiconesculentum Lyc e 1 profilin 14 C AJ417553 tomato Lyc e 2b-fructofuranosidase 50 C see isoallergen list Lyc e 3 lipid transferprot. 6 C U81996 Solanum tuberosum Sola t 1 patatin 43 P P15476 potatoSola t 2 cathepsin D inhibitor 21 P P16348 Sola t 3 cysteine proteaseinhibitor 21 P P20347 Sola t 4 aspartic protease inhibitor 16 + 4 PP30941 Bertholletia excelsa Ber e 1 2S albumin  9 C P04403, M17146Brazil nut Ber e 2 11S globulin seed storage protein 29 C AY221641Juglans nigra Jug n 1 2S albumin  19* C AY102930 black walnut Jug n 2vicilin-like prot.  56* C AY102931 Juglans regia Jug r 1 2S albumin CU66866 English walnut Jug r 2 vicilin 44 C AF066055 Jug r 3 lipidtransfer protein  9 P Pastorello Anacardium occidentale Ana o 1vicilin-like protein 50 C see isoallergen list Cashew Ana o 2legumin-like protein 55 C AF453947 Ana o 3 2S albumin 14 C AY081853Ricinus communis Ric c 1 2S albumin C P01089 Castor bean Sesamum indicumSes i 1 2S albumin  9 C 121A, AF240005 sesame Ses i 2 2S albumin  7 CAF091841 Ses i 3 7S vicilin-like globulin 45 C AF240006 Ses i 4 oleosin17 C AAG23840 Ses i 5 oleosin 15 C AAD42942 Cucumis melo Cuc m 1 serineprotease 66 C D32206 muskmelon Cuc m 2 profilin 14 C AY271295 Cuc m 3pathogenesis-rel p. PR-1  16* P P83834 I. Others Ani s 1 24 P 121B,A59069 Anisakis simplex Ani s 2 paramyosin 97 C AF173004 nematode Ani s3 tropomyosin 41 C 121C, Y19221 Ani s 4  9 P P83885 Argas reflexus Arg r1 17 C AJ697694 piigeon tick Ascaris suum Asc s 1 10 P 122 worm Caricapapaya Car p 3w papain   23.4* C 122A, M15203 papaya Dendronephthyanipponica Den n 1 53 P 122B soft coral Hevea brasiliensis Hev b 1elongation factor 58 P 123, 124 rubber (latex) Hev b 2 1,3-glucanase34/36 C 125 Hev b 3 24 P 126, 127 Hev b 4 component of 100-115 P 128microhelix complex Hev b 5 16 C U42640 Hev b 6.01 hevein precursor 20 CM36986, p02877 Hev b 6.02 hevein  5 C M36986, p02877 Hev b 6.03C-terminal fragment 14 C M36986, p02877 Hev b 7.01 hom: patatin fromB-serum 42 C U80598 Hev b 7.02 hom: patatin from C-serum 44 C AJ223038Hev b 8 profilin 14 C see list of isoallergens Hev b 9 enolase 51 CAJ132580 Hev b 10 Mn superoxide dismut. 26 C see list of isoallergensHev b 11 class 1 chitinase C see list of isoallergens Hev b 12 lipidtransfer protein   9.3 C AY057860 Hev b 13 esterase 42 P P83269 Homosapiens Hom s 1  73* C Y14314 human autoallergens Hom s 2   10.3* CX80909 Hom s 3   20.1* C X89985 Hom s 4  36* C Y17711 Hom s 5   42.6* CP02538 Triplochiton scleroxylon Trip s 1 class 1 chitinase   38.5 PKespohl p.c. obeche

REFERENCES

-   1 Marsh, D. G., and L. R. Freidhoff. 1992. ALBE, an allergen    database. IUTS, Baltimore, Md., Edition 1.0.-   2 Marsh, D. G. et al. 1986. Allergen nomenclature. Bull WHO    64:767-770.-   3 King, T. P. et al. 1964. Biochemistry 3:458-468.-   4 Lowenstein, H. 1980. Allergy 35:188-191.-   5 Aukrust, L. 1980. Allergy 35:206-207.-   6 Demerec, M. et al. 1966. Genetics 54:61-75.-   7 Bodmer, J. G. et al. 1991. Immunogenetics 33:301-309.-   8 Griffith, I. J. et al. 1991. Int. Arch. Allergy Appl. Immunol.    96:296-304.-   9 Roebber, M. et al. 1985. J. Immunol. 134:3062-3069.-   10 Metzler, W. J. et al. 1992. Biochemistry 31:5117-5127.-   11 Metzler, W. J. et al. 1992. Biochemistry 31:8697-8705.-   12 Goodfriend, L. et al. 1979. Fed. Proc. 38:1415.-   13 Ekramoddoullah, A. K. M. et al. 1982. Mol. Immunol. 19:1527-1534.-   14 Ansari, A. A. et al. 1987. J. Allergy Clin. Immunol. 80:229-235.-   15 Morgenstern, J. P. et al. 1991. Proc. Natl. Acad. Sci. USA    88:9690-9694.-   16 Griffith, I. J. et al. 1992. Gene 113:263-268.-   17 Weber, A. et al. 1986. Biochem. Physiol. 83B:321-324.-   18 Weber, A. et al. 1987. Allergy 42:464-470.-   19 Stanworth, D. R. et al. 1990. Bulletin WHO 68:109-111.-   20 Rafnar, T. et al. 1991. J. Biol. Chem. 266: 1229-1236.-   21 Rogers, B. L. et al. 1991. J. Immunol. 147:2547-2552.-   22 Klapper, D. G. et al. 1980. Biochemistry 19:5729-5734.-   23 Ghosh, B. et al. 1993. J. Immunol. 150:5391-5399.-   24 Roebber, M. et al. 1983. J. Immunol. 131:706-711.-   25 Lubahn, B., and D. G. Klapper. 1993. J. Allergy Clin. Immunol.    91:338.-   26 Roebber, M., and D. G. Marsh. 1991. J. Allergy Clin. Immunol.    87:324.-   27 Goodfriend L. et al. Mol Immunol 22: 899-906, 1985.-   28 Himly M. et al. FASEB J 17: 106-108, 2003.-   28A Nilsen, B. M. et al. 1991. J. Biol. Chem. 266:2660-2668.-   29 Wopfner N. et al. Biol Chem 383: 1779-1789, 2002.-   29A Jimenez A. et al. 1994. Int Arch Allergy Immunol 105:297-307.-   29B Barderas R. et al. Int Arch Allergy Immunol 127: 47-54, 2002.-   29C Carnés J. et al. Allergy 56, Supplement 68: 274, 2001.-   29D Giuliani A. et al. Allergy 42: 434-440, 1987.-   30 Smith, P. M. et al. 1996. J. Allergy Clin. Immunol. 98:331-343.-   31 Suphioglu, C. et al. 1997. FEBS Left. 402:167-172.-   31a Asturias J. A. et al. 1997. Clin Exp Allergy 27:1307-1313.-   32 Mecheri, S. et al. 1985. Allergy Appl. Immunol. 78:283-289.-   33 Roberts, A. M. et al. 1993. Allergy 48:615-623.-   33a Guerin-Marchand, C. et al. 1996. Mol. Immunol. 33:797-806.-   34 Klysner, S. et al. 1992. Clin. Exp. Allergy 22: 491-497.-   35 Perez, M. et al. 1990. J. Biol. Chem. 265:16210-16215.-   36 Griffith, I. J. et al. 1991. FEBS Letters 279:210-215.-   37 Ansari, A. A. et al. 1989. J. Biol. Chem. 264:11181-11185.-   37a Sidoli, A. et al. 1993. J. Biol. Chem. 268:21819-21825.-   38 Ansari, A. A. et al. 1989. Biochemistry 28:8665-8670.-   39 Singh, M. B. et al. 1991. Proc. Natl. Acad. Sci. 88:1384-1388.-   39a van Ree R. et al. 1995. J Allergy Clin Immunol 95:970-978.-   40 Suphioglu, C. and Singh, M. B. 1995. Clin. Exp. Allergy    25:853-865.-   41 Dolecek, C. et al. 1993. FEBS Lett. 335:299-304.-   41A Fischer S. et al. 1996. J Allergy Clin Immunol 98:189-198.-   42 Matthiesen, F., and H. Lowenstein. 1991. Clin. Exp. Allergy    21:297-307.-   43 Petersen, A. et al. 1995. Int. Arch. Allergy Immunol. 108:55-59.-   43A Marknell DeWitt A. et al. Clin Exp Allergy 32: 1329-1340, 2002.-   44 Valenta, R. et al. 1994. Biochem. Biophys. Res. Commun.    199:106-118.-   46 Esch, R. E., and D. G. Klapper. 1989. Mol. Immunol. 26:557-561.-   47 Olsen, E. et al. 1991. J. Immunol. 147:205-211.-   48 Avjioglu, A. et al. 1993. J. Allergy Clin. Immunol. 91:340.-   52 Kos T. et al. 1993. Biochem Biophys Res Commun 196:1086-92.-   53 Díaz-Perales A. et al. 2000. Clin Exp Allergy 30:1403-1410.-   54 Ipsen, H., and O. C. Hansen. 1991. Mol. Immunol. 28: 1279-1288.-   55 Taniai, M. et al. 1988. FEBS Lett. 239:329-332.-   56 Griffith, I. J. et al. 1993. J. Allergy Clin. Immunol. 91:339.-   57 Sakaguchi, M. et al. Allergy 45: 309-312, 1990.-   57A Yokoyama M. et al. Biochem Biophys Res Commun 275: 195-202,    2000.-   57B Midoro-Horiuti T. et al. J Immunol 164: 2188-2192, 2000.-   57C Tinghino R. et al. J. Allergy Clin. Immunol. 101: 772-777, 1998.-   58 Gross G N et al. Scand J Immunol 8: 437-441, 1978.-   58A Obispo T M et al. Clin Exp Allergy 23: 311-316, 1993.-   58B Midoro-Horiuti T. et al. Clin Exp Allergy 31: 771-778, 2001.-   59 Lombardero M. et al. Clin. Exp. Allergy 24: 765-770, 1994.-   60 Villalba, M. et al. Eur. J. Biochem. 216: 863-869, 1993.-   60A Asturias J A et al. J Allergy Clin Immunol 100: 365-372, 1997.-   60B Batanero E. et al. Eur J Biochem 241: 772-778, 1996.-   60C Batanero E. et al. FEBS Lett. 410: 293-296, 1997.-   60D Tejera M L et al. J Allergy Clin Immunol 104: 797-802, 1999.-   60E Ledesma A. et al. FEBS Lett 466: 192-196, 2000.-   60F Barral P. et al. J Immunol 172: 3644-3651, 2004.-   61 Yi F C et al. Clin Exp Allergy 32: 1203-1210, 2002.-   61A Ramos J D et al. Int Arch Allergy Immunol 126: 286-293, 2001.-   62 Chua, K. Y. et al. J. Exp. Med. 167: 175-182, 1988.-   62A Chua, K. Y. et al. Int. Arch. Allergy Appl. Immunol. 91:    118-123, 1990.-   62B Smith A M et al. Int Arch Allergy Immunol 124: 61-63, 2001.-   62C Smith A M et al. J Allergy Clin Immunol 107: 977-984, 2001.-   63 Smith W A, Thomas W R. Int Arch Allergy Immunol 109: 133-140,    1996.-   64 Lake, F. R. et al. J. Allergy Clin. Immunol. 87: 1035-1042, 1991.-   65 Tovey, E. R. et al. J. Exp. Med. 170: 1457-1462, 1989.-   66 Yasueda, H., T. Shida, T. Ando, S. Sugiyama, and H.    Yamakawa. 1991. Allergenic and proteolytic properties of fourth    allergens from Dermatophagoides mites. In: “Dust Mite Allergens and    Asthma. Report of the 2nd international workshop” A. Todt, Ed., UCB    Institute of Allergy, Brussels, Belgium, pp. 63-64.-   67 Shen, H.-D. et al. Clin. Exp. Allergy 23: 934-940, 1993.-   67A O'Neil G M et al. Biochim Biophys Acta, 1219: 521-528, 1994.-   67B King C. et al. J Allergy Clin Immunol 98: 739-747, 1996.-   68 Lind P. et al. J. Immunol. 140: 4256-4262, 1988.-   69 Dilworth, R. J. et al. Clin. Exp. Allergy 21: 25-32, 1991.-   70 Nishiyama, C. et al. Int. Arch. Allergy Immunol. 101: 159-166,    1993.-   70A Trudinger, M. et al. Clin. Exp. Allergy 21: 33-38, 1991.-   71 Shen H D et al. Clin Exp Allergy 25: 1000-1006, 1995.-   71A Tategaki A. et al. ACI International suppl. 1: 74-76, 2000.-   72 Aki T. et al. J Allergy Clin Immunol 96: 74-83, 1995.-   72A Tsai L. et al. Clin Exp Allergy 29: 1606-1613, 1999.-   72B Gafvelin G. et al. J Allergy Clin Immunol 107: 511-518, 2001.-   73 van Hage-Hamsten. et al. J. Allergy Clin. Immunol. 91:353, 1993.-   74 Varela J. et al. Eur J Biochem 225: 93-98, 1994.-   74A Schmidt M. et al. FEBS Lett 370: 11-14, 1995.-   75 Eriksson T L J et al. Eur. J. Biochem. 268: 287-294, 2001.-   75A Saarne T. et al. Int Arch Allergy Immunol 130: 258-265, 2003.-   75B Eriksson T L et al. Eur. J. Biochem. 251 (1-2), 443-447, 1998.-   76 Rautiainen J, Rytkonen M, Pelkonen J, Pentikainen J, Perola O,    Virtanen T, Zeiler T, Mantyjarvi R. BDA20, a major bovine dander    allergen characterised at the sequence level is Bos d 2. Submitted.-   77 Gjesing B, Lowenstein H. Ann Allergy 53:602, 1984.-   78 de Groot, H. et al. J. Allergy Clin. Immunol. 87:1056-1065, 1991.-   79 Konieczny, A. Personal communication; Immunologic Pharmaceutical    Corp.-   79A Bulone, V. Eur J Biochem 253: 202-211, 1998.-   79B Swiss-Prot acc. P81216, P81217.-   79C Dandeu J. P. et al. (1993). J. Chromatogr. 621:23-31.-   79D Goubran Botros H. et al. 1998. J. Chromatogr. B 710:57-65.-   79E Hilger C. et al. Allergy 52: 179-187; and Hilger C. et al. Gene    169:295-296, 1996.-   79F Ichikawa K. et al. Clin Exp Allergy, In Press 2001.-   80 Fahlbusch B. et al. Allergy 57: 417-422, 2002.-   81 McDonald, B. et al. 1988. J. Allergy Clin. Immunol. 83:251.-   81A Clarke, A. J. et al. 1984. EMBO J 3:1045-1052.-   82 Longbottom, J. L. 1983. Characterisation of allergens from the    urines of experimental animals. McMillan Press, London, pp. 525-529.-   83 Laperche, Y. et al. 1983. Cell 32:453-460.-   83A Bush R K et al. 1999. J Allergy Clin Immunol 104:665-671.-   83B Aukrust L, Borch S M. 1979. Int Arch Allergy Appl Immunol    60:68-79.-   83C Sward-Nordmo M. et al. 1988. Int Arch Allergy Appl Immunol    85:288-294.-   84 Shen, et al. J. Allergy Clin. Immunol. 103:S157, 1999.-   84A Crameri R. Epidemiology and molecular basis of the involvement    of Aspergillus fumigatus in allergic diseases. Contrib. Microbiol.    Vol. 2, Karger, Basel (in press).-   84B Shen, et al. (manuscript submitted), 1999-   84C Shen H D et al. Vacuolar serine proteinase: A major allergen of    Aspergillus fumigatus. 10th International Congress of Immunology,    Abstract, 1998.-   85 Kumar A. et al. 1993. J. Allergy Clin. Immunol. 91:1024-1030.-   85A Saxena S. et al. 2003. Clin Exp Immunol 134:86-91.-   85B Baur X. et al. Allergy 57: 943-945, 2002.-   86A Shen H D et al. 1996. Clin Exp Allergy 26:444-451.-   86B Shen, et al. Abstract; The XVIII Congress of the European    Academy of Allergology and Clinical Immunology, Brussels, Belgium,    3-7 Jul. 1999.-   87 Shen H D et al. Clin Exp Allergy 29: 642-651, 1999.-   87A Shen H D et al. Clin Exp Allergy 25: 350-356, 1995.-   87B Shen H D et al. J Lab Clin Med 137: 115-124, 2001.-   88 Woodfolk J A et al. 1998. J Biol Chem 273:29489-96.-   88A Deuell, B. et al. 1991. J. Immunol. 147:96-101.-   89 Shen, H. D. et al. 1991. Clin. Exp. Allergy 21:675-681.-   89A Horner W E et al. 1995. Int Arch Allergy Immunol 107:298-300.-   89B Chang C Y et al. J Biomed Sci 9: 645-655, 2002.-   90 Yasueda H. et al. Biochem Biophys Res Commun 248: 240-244, 1998.    NB:strain TIMM2782 (Teikyo University Institute for Medical    Mycology) equal to strain CBS1878 (Central Bureau von    Schimmelkulturen).-   90A Onishi Y. et al. Eur J Biochem 261: 148-154, 1999. NB: strain    TIMM2782 (Teikyo University Institute for Medical Mycology) equal to    strain CBS1878 (Central Bureau von Schimmelkulturen).-   91 Schmidt M. et al. Eur J Biochem 246:181-185, 1997. NB: strain    ATCC no. 42132 (American Type Culture Collection).-   91A Rasool O. et al. Eur J Biochem 267: 4355-4361, 2000. NB: strain    ATCC no. 42132 (American Type Culture Collection).-   91B NB: strain 4625 (Indian Agricultural Research Institute, PUSA;    New Delhi, India).-   92 Kuchler, K. et al. 1989. Eur. J. Biochem. 184:249-254.-   93 Gmachl, M., and G. Kreil. 1993. Proc. Natl. Acad. Sci. USA    90:3569-3573.-   93A Hoffman D R. 1977. J Allergy Clin. Immunol. 59:364-366.-   94 Habermann, E. 1972. Science 177:314-322.-   95 Hoffman D R, Jacobson R S. 1996. J. Allergy Clin. Immunol.    97:812-821.-   95A Hoffman D R, El-Choufani A E, Smith M M, de Groot H. 2001.    Occupational allergy to bumblebee venom: Allergens of Bombus    terrestris. J Allergy Clin Immunol In press.-   95B Helm R. et al. 1996. J Allerg Clin Immunol 98:172-180.-   95C Pomes A. et al. 1998. J Biol Chem 273:30801-30807.-   96 Arruda L K et al. J Biol Chem 270:19563-19568, 1995.-   97 Arruda L K et al. J Biol Chem 270:31196-31201, 1995.-   98 Arruda L K et al. Int Arch Allergy Immunol 107:295-297, 1995.-   98A Wu C H et al. 1998. J Allergy Clin Immunol 101:832-840.-   98B Melen E. et al. 1999. J Allergy Clin Immunol 103:859-64.-   98C Wu C H et al. J Biol Chem 271:17937-17943, 1996.-   98D Wu C H et al. Molecular Immunol 34:1-8, 1997.-   98E Santos A B R et al. 1999. J Allergy Clin Immunol 104:329-337.-   98F Asturias J A et al. 1999. J Immunol 162:4342-4348.-   99 Mazur, G. et al. 1990. Monog. Allergy 28:121-137.-   99A Moneo I. et al. Allergy 58: 34-37, 2003.-   100 Soldatova, L. et al. 1993. FEBS Letters 320:145-149.-   101 Lu, G. et al. 1994. J. Allergy Clin. Immunol. 93:224.-   102 Fang, K. S. F. et al. 1988. Proc. Natl. Acad. Sci., USA    85:895-899.-   103 King, T. P. et al. 1990. Prot. Seq. Data Anal. 3:263-266.-   104 Lu, G. et al. 1993. J. Immunol. 150: 2823-2830.-   105 King, T. P. and Lu, G. 1997. Unpublished data.-   105A King T P et al. 1996. J. Allergy Clin. Immunol. 98:588-600.-   106 Hoffman, D. R. 1993. J. Allergy Clin. Immunol. 92:707-716.-   107 Hoffman D R. 1992. Unpublished data.-   108 Hoffman D R. J. Allergy Clin. Immunol. 91:187, 1993.-   109 Jacobson R S et al. J. Allergy Clin. Immunol. 89:292, 1992.-   110 Hoffman D R. J. Allergy Clin. Immunol 91: 71-78, 1993.-   111 Schmidt M. et al. FEBS Letters 319: 138-140, 1993.-   111A Paddock C D et al. J Immunol 167: 2694-2699, 2001.-   112 Elsayed S, Bennich H. Scand J Immunol 3: 683-686, 1974.-   113 Elsayed S. et al. Immunochemistry 9: 647-661, 1972.-   114 Hoffman, D. R. 1983. J. Allergy Clin. Immunol. 71: 481-486.-   115 Langeland, T. 1983. Allergy 38:493-500.-   116 Daul C B, Slattery M, Morgan J E, Lehrer S B. 1993. Common    crustacea allergens: identification of B cell epitopes with the    shrimp specific monoclonal antibodies. In: “Molecular Biology and    Immunology of Allergens” (D. Kraft and A. Sehon, eds.). CRC Press,    Boca Raton. pp. 291-293.-   116A Shanti K N et al. J. Immunol. 151: 5354-5363, 1993.-   117 Yu C J et al. J Immunol 170: 445-453, 2003.-   117A Miyazawa M et al. J. Allergy Clin. Immunol. 98: 948-953, 1996.-   117B Asturias J A et al. Int Arch Allergy Immunol 128: 90-96, 2002.-   117C Lopata A L et al. J. Allergy Clin. Immunol. 100: 642-648, 1997.-   117D Hoffmann-Sommergruber K. et al. Clin. Exp. Allergy 29: 840-847,    1999.-   118 Monsalve R I et al. Biochem. J. 293: 625-632 1993.-   118A. Monsalve R I et al. 1997. Clin Exp Allergy 27:833-841.-   119 Mena, M. et al. Plant Molec. Biol. 20: 451-458, 1992.-   119A Palosuo K. et al. J. Allergy Clin. Immunol. 108: 634-638, 2001.-   119B Xu H. et al. Gene 164: 255-259, 1995.-   119C Pastorello E A et al. J. Allergy Clin. Immunol. 94: 699-707,    1994.-   119D Diaz-Perales A. et al. J Allergy Clin Immunol 110: 790-796,    2002.-   119E Galleguillos F, Rodriguez J C. Clin Allergy 8: 21-24, 1978.-   119F Baur X. Clin Allergy 9: 451-457, 1979.-   119G Gailhofer G. et al. Clin Allergy 18: 445-450, 1988.-   120 Menendez-Arias, L. et al. 1988. Eur. J. Biochem. 177:159-166.-   120A Gonzalez R. et al. Lancet 346:48-49, 1995.-   120B Kleine-Tebbe J. et al. J Allergy Clin Immunol 110: 797-804,    2002.-   120C Sanchez-Monge R. et al. J. Allergy Clin. Immunol. 106: 955-961,    2000.-   121 Gavrovic-Jankulovic M. et al. J Allergy Clin Immun of 110:    805-810, 2002.-   121A Pastorello E A et al. J. Chromatogr. B Biomed. Sci. Appl. 756:    85-93, 2001.-   121B Moneo I. et al. J. Allergy Clin. Immunol. 106: 177-182, 2000.-   121C Asturias J A et al. 2000. Allergy 55:898-890.-   122 Christie, J. F. et al. 1990. Immunology 69:596-602.-   122A Baur X. et al. Clin Allergy 12: 9-17, 1982.-   122B Onisuka R. et al. Int Arch Allergy Immunol 125: 135-143, 2001.-   123 Czuppon A B et al. J Allergy Clin Immunol 92:690-697, 1993.-   124 Attanayaka DPSTG et al. 1991. Plant Mol Biol 16:1079-1081.-   125 Chye M L, Cheung K Y. 1995. Plant Mol Biol 26:397-402.-   126 Alenius H. et al. 1993. Int Arch Allergy Immunol 102:61-66.-   127 Yeang H Y, Cheong K F, Sunderasan E, Hamzah S, Chew N P, Hamid    S, Hamilton R G, Cardosa M J. 1996. The 14.6 kD (REF, Hey b 1) and    24 kD (Hey b 3) rubber particle proteins are recognised by IgE from    Spina Bifida patients with Latex allergy. J Allerg Clin Immunol in    press.-   128 Sunderasan E. et al. 1995. J nat Rubb Res 10:82-99.-   129 Swoboda I. et al. 2002. J Immunol. 168:4576-84.-   130 Vrtala et al., 2007. J Immunol. 179:1731-1739.-   131 Valenta and Niederberger, 2007. J Allergy Clin Immunol.    119(4):826-830.

According to a particularly preferred embodiment of the presentinvention at least one, preferably at least two, more preferably atleast three, in particular all, of the at least three peptides derivedfrom the at least one wild-type allergen is a B cell binding peptide.

“B cell binding peptides” to be used for allergy vaccination accordingto the invention are derived from or close to the IgE binding sites ofallergens but per se show no or minimal IgE reactivity compared to thewild-type allergen (Focke M et al. Clinical & Experimental Allergy40(2010):385-397). Requirements for their production and selection arethe knowledge of the primary sequence of the allergen and regarding theIgE binding sites. Upon immunization, B cell binding peptides fused to asuitable immunogenic carrier, are capable of inducing the production ofallergen-specific IgG which can block IgE binding to the allergen.Whether the IgG induced with the fusion protein can recognize theallergen can be determined, for instance, by testing the IgG forreactivity with the complete allergen. Suitable methods include ELISA,dot blot or Western blot assays. Those peptides are preferred whichinduce IgG that blocks patients IgE binding to the allergen.

The present invention shows that the use of suitable B cell bindingpeptides in particular when three or more are fused to a suitablecarrier according to the present invention allows the induction of IgGresponses which are better focused to the IgE epitopes than thoseinduced by immunization even with a complete allergen. Furthermore, theinvention shows that the combination of the appropriate peptides andtheir number with a suitable carrier can direct the allergen-specificimmune response towards a favorable anti-allergic immune response(characterized by the induction of preferentially allergen-specific IgGand not IgE responses and tolerogenic (IL-10) and Th1 (Interferon gamma)cytokine responses.

Moreover, it surprisingly turned out that—despite the fact that theylack allergen-specific T-cell epitopes—polypeptides according to theinvention containing 3 or more B cell binding peptides fused to animmunogenic carrier are able reduce allergen-specific T-cell reactions.This is shown by the fact that the presence of allergen-specific IgGinduced by therapeutic vaccination with the hypoallergenic polypeptidesof the present invention reduces allergen-specific T-cell activationcaused by IgE facilitated antigen presentation in PBMCs from vaccinatedhuman allergic individuals. (FIGS. 16 A and B).

According to a preferred embodiment of the present invention at leastone of said at least three peptides exhibits no or reduced IgE-bindingcapacity compared to the wild-type allergen.

According to another preferred embodiment of the present invention atleast one, preferably at least two, more preferably at least three, ofsaid at least three B-cell binding peptides exhibits no or substantiallyno T-cell reactivity.

The presence of allergen-specific T cell epitopes may give rise tounwanted T cell mediated side effects. Therefore it is particularlypreferred to use peptides exhibiting no or substantially no T-cellreactivity in order to obtain the polypeptides of the present invention.

However, also allergen fragments comprising at least one T-cell epitopemay be used in the polypeptide according to the present invention.

“Exhibiting reduced IgE-binding capacity”, as used herein, means thatthe molecules according to the present invention show significantlyreduced IgE-binding capacity or activity (at least 50% less, preferablyat least 70% less, more preferably at least 80% less, even morepreferably at least 90% less, most preferably at least 95% less, bindingcapacity compared to the wild-type allergen) or even lack IgE-binding atall.

IgE-binding activity/capacity of molecules like peptides and proteinscan be determined by, for example, an enzyme linked immunosorbent assay(ELISA) using, for example, sera obtained from a subject, (i.e., anallergic subject) that has been previously exposed to the wild-typeallergen. Briefly, a peptide to be tested is coated onto wells of amicrotiter plate. After washing and blocking the wells, an antibodysolution consisting of the plasma of an allergic subject, who has beenexposed to the peptide being tested or the protein from which it wasderived, is incubated in the wells. A labelled secondary antibody isadded to the wells and incubated. The amount of IgE-binding is thenquantified and compared to the amount of IgE bound by a purifiedwild-type allergen.

Alternatively, the binding activity of a peptide can be determined byWestern blot analysis. For example, a peptide to be tested is run on apolyacrylamide gel using SDS-PAGE. The peptide is then transferred tonitrocellulose and subsequently incubated with serum from an allergicsubject. After incubation with the labelled secondary antibody, theamount of IgE bound is determined and quantified.

Another assay which can be used to determine IgE-binding activity of apeptide is a competition ELISA assay. Briefly, an IgE-antibody pool isgenerated by combining plasma from allergic subjects who have been shownby direct ELISA to be IgE-reactive with wild-type allergen. This pool isused in ELISA competition assays to compare IgE-binding to wild-typeallergen to the peptide tested. IgE-binding for the wild-type allergenand the peptide being tested is determined and quantified.

A “T-cell epitope” means a protein, peptide or polypeptide (e.g.,allergen) or fragment thereof, for which a T-cell has an antigenspecific binding site, the result of binding to said binding siteactivates the T-cell. The term “exhibiting reduced T-cell reactivity”,as used herein, refers to molecules which exhibit a T-cell reactivitywhich is significantly reduced compared to the stimulation induced bythe wild-type allergen from which the hypoallergenic molecule isderivedusing equimolar amounts in standard assays known in the art(reduced T-cell reactivity means at least 30%, preferably at least 50%,more preferably at least 70%, most preferably at least 90%, lessstimulation of hypoallergenic molecules compared to the wildtypeallergen at equimolar amounts). In a particular preferred embodiment ofthis invention, the molecules may “lack” T-cell epitopes and thusmolecule shows reduced T-cell reactivity in the individual(s) to betreated (i.e., who is to receive an epitope-presenting valency platformmolecule). It is likely that, for example, an allergen-derived moleculemay lack a T-cell epitope(s) with respect to an individual, or a groupof individuals, while possessing a T-cell epitope(s) with respect toother individual(s). Methods for detecting the presence of a T-cellepitope are known in the art and include assays which detect T-cellproliferation (such as thymidine incorporation). Immunogens that fail toinduce statistically significant incorporation of thymidine abovebackground (i.e., generally p less than 0.05 using standardstatistically methods) are generally considered to lack T-cell epitopes,although it will be appreciated that the quantitative amount ofthymidine incorporation may vary, depending on the immunogen beingtested (see, e.g., Zhen L. et al. (Infect Immun. (2003) 71:3920-3926)).Generally, a stimulation index below about 2-3, more preferably lessthan about 1, indicates lack of T-cell reactivity and epitopes. Thepresence of T-cell epitopes can also be determined by measuringsecretion of T-cell-derived lymphokines according to standard methods.The stimulation index (SI) may be calculated by dividing theproliferation rate (Thymidine uptake) of stimulated cells through theproliferation rate of unstimulated cells in medium alone. SI=1 means nostimulation, and SI>1 indicates stimulation of cells. Location andcontent of T-cell epitopes, if present, can be determined empirically.

The cytokine secretion may be determined in addition to the stimulationof T cells. For example, IFN-gamma and IL-10 as biomarkers for increasedactivity of regulatory T cells have been recognized as cytokinesaccompanying a successful allergy immunotherapy.

The peptide fragments of the present invention are preferably composedor consist of amino acids 151 to 177, 87 to 117, 1 to 30, 43 to 70 or212 to 241 of Phl p 1, amino acids 1 to 33, 8 to 39, 34 to 65 or 66 to96 of Phl p 2, amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58, 132to 162, 217 to 246, 252 to 283 or 176 to 212 of Phl p 5, amino acids 23to 54, 56 to 90, 73 to 114 or 95 to 127 of Phl p 6, amino acids 1 to 34or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64to 92 of chain 2 of Fel d 1, amino acids 30 to 59, 50 to 79, 75 to 104,30 to 74 or 60 to 104 of Bet v 1, amino acids 1 to 30, 52 to 84 or 188to 222 of Der p 1, amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1-35, 36-70,71-110, 111-145, 140-170, 175-205, 210-250 or 250-284 of Der p 10, aminoacids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys->Ser)of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 ofAlt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 ofOle e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200,225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80,81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variationsthereof.

The specific amino acid sequences of the above identifiedallergen-derived molecules are (peptides in the following table havingan N- and/or C-terminal cysteine residue (C) being used in thepolypeptide of the present invention may lack said cysteine residue):

Peptide Position Sequence SEQ ID No. Pep Alt a 1.1 19-58APLESRQDTASCPVTTEGDYVWKISEFYGRKPEGTYYN 23 SL Pep Alt a 1.2 59-95GFNIKATNGGTLDFTCSAQADKLEDHKWYSCGENSFM 24 Pep Alt a 1.3  91-120ENSFMDFSFDSDRSGLLLKQKVSDDITYVA 25 Pep Alt a 1.4 121-157 TATLPNYCRAGGNGPKDFVCQGVADAYITLVTLPKSS 26 Pep Alt a 2.1  1-40MHSSNNFFKDNIFRSLSKEDPDYSRNIEGQVIRLHWDW 27 AQ Pep Alt a 2.2 41-80LLMLSAKRMKVAFKLDIEKDQRVWDRCTADDLKGRN 28 GFKR Pep Alt a 2.3  81-120CLQFTLYRPRDLLSLLNEAFFSAFRENRETIINTDLEYAA 29 Pep Alt a 2.4 121-160KSISMARLEDLWKEYQKIFPSIQVITSAFRSIEPELTVYT 30 Pep Alt a 2.5 161-190CLKKIEASFELIEENGDPKITSEIQLLKAS 31 Pep Alt a 6.1  1-34MTITKIHARSVYDSRGNPTVEVDIVTETGLHRAI 32 Pep Alt a 6.2 35-74VTETGLHRAIVPSGASTGSHEACELRDGDKSKWGGKGV 33 TK Pep Alt a 6.3  74-115APALIKEKLDVKDQSAVDAFLNKLDGTTNKTNLGANAI 34 LGVS Pep Alt a 6.4 125-165EKGVPLYAHISDLAGTKKPYVLPVPF 35 QNVLNGGSHAGGRLA Pep Alt a 6.5 174-213CEAPTFSEAMRQGAEVYQKLKALAKKTYGQSAGNVGD 36 EGG Pep Alt a 6.6 241-280IKIAMDVASSEFYKADEKKYDLDFKNPDSDKSKWLTYE 37 QL Pep Alt a 6.7 294-333VSIEDPFAEDDWEAWSYFFKTYDGQIVGDDLTVTNPEFI 38 K Pep Alt a 6.8 361-400AKDAFGAGWGVMVSHRSGETEDVTIADIVVGLRSGQIK 39 TG Pep Alt a 6.9 401-438APARSERLAKLNQILRIEEELGDNAVYAGNNFRTAVNL 40 Pep Amb a 1.1 31-70EILPVNETRRLTTSGAYNIIDGCWRGKADWAENRKALA 41 DC Pep Amb a 1.2  80-120GGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNRPLWI 42 IFE Pep Amb a 1.3 125-155IRLDKEMVVNSDKTIDGRGAKVEIINAGFTL 43 Pep Amb a 1.4 160-200NVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDGDAIS 44 IS Pep Amb a 1.5 225-263GTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLATVA 45 F Pep Amb a 1.6 264-300NTFTDNVDQRMPRCRHGFFQVVNNNYDKWGSYAIGGS 46 Pep Amb a 1.7 305-350ILSQGNRFCAPDERSKKNVLGRHGEAAAESMKWNWRT 47 NKDVLENGA Pep Amb a 1.8 356-396GVDPVLTPEQSAGMIPAEPGESALSLTSSAGVLSCQPGA 48 PC Pep Art v 1.1 27-70SKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHK 49 REAGKES Pep Art v 1.2  70-100SCFCYFDCSKSPPGATPAPPGAAPPPAAGGS 50 Pep Art v 1.3  92-132APPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPST 51 H Can f 1 Pep 1 19-56QDTPALGKDTVAVSGKWYLKAMTADQEVPEKPDSVTP 52 M Can f 1 Pep 2 51-90DSVTPMILKAQKGGNLEAKITMLTNGQCQNITVVLHKT 53 SE Can f 1 Pep 3  78-118CQNITVVLHKTSEPGKYTAYEGQRVVFIQPSPVRDHYIL 54 YC Can f 1 Pep 4 106-145QPSPVRDHYILYCEGELHGRQIRMAKLLGRDPEQSQEA 55 LE Can f 1 Pep 5 135-174RDPEQSQEALEDFREFSRAKGLNQEILELAQSETCSPGG 56 Q Can f 2 Pep 1 19-58QEGNHEEPQGGLEELSGRWHSVALASNKSDLIKPWGHF 57 RV Can f 2 Pep 2 52-91PWGHFRVFIHSMSAKDGNLHGDILIPQDGQCEKVSLTAF 58 K Can f 2 Pep 3  82-119CEKVSLTAFKTATSNKFDLEYWGHNDLYLAEVDPKSYL 59 Can f 2 Pep 4 106-144NDLYLAEVDPKSYLILYMINQYNDDTSLVAHLMVRDLS 60 R Can f 2 Pep 5 139-180VRDLSRQQDFLPAFESVCEDIGLHKDQIVVLSDDDRCQ 61 GSRD Fel d 2 Pep 1 25-58EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQC 62 Fel d 2 Pep 2  99-133CTVASLRDKYGEMADCCEKKEPERNECFLQHKDDN 63 Fel d 2 Pep 3 154-183NEQRFLGKYLYEIARRHPYFYAPELLYYAE 64 Fel d 2 Pep 4 277-307CADDRADLAKYICENQDSISTKLKECCGKPV 65 Fel d 2 Pep 5 334-363VEDKEVCKNYQEAKDVFLGTFLYEYSRRHP 66 Fel d 2 Pep 6 373-402LAKEYEATLEKCCATDDPPACYAHVFDEFK 67 Fel d 2 Pep 7 544-573EKQIKKQSALVELLKHKPKATEEQLKTVMG 68 Fel d 2 Pep 8 579-608VDKCCAAEDKEACFAEEGPKLVAAAQAALA 69 Fel d 2 Pep 9 58-99CPFEDHVKLVNEVTEFAKGCVADQSAANCEKSLHELLG 70 DKLC Fel d 2 Pep 10 125-165CFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLGK 71 YLYE Fel d 2 Pep 11 183-224EEYKGVFTECCEAADKAACLTPKVDALREKVLASSAKE 72 RLKC Fel d 2 Pep 12 224-261CASLQKFGERAFKAWSVARLSQKFPKAEFAEISKLVTD 73 Fel d 2 Pep 13 252-289FAEISKLVTDLAKIHKECCHGDLLECADDRADLAKYIC 74 Fel d 2 Pep 14 303-340CGKPVLEKSHCISEVERDELPADLPPLAVDFVEDKEVC 75 Fel d 2 Pep 15 416-457CELFEKLGEYGFQNALLVRYTKKVPQVSTPTLVEVSRSL 76 GKV Fel d 2 Pep 16 460-500 CTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSERVTK 77 C Fel d 2 Pep 17 501-542 CTESLVNRRPCFSALQVDETYVPKEFSAETFTFHADLCT 78 LPE Pep Ole e 1.1  1-40EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIPGASLR 79 Pep Ole e 1.2 36-66GASLRLQCKDKENGDVTFTEVGYTRAEGLYS 80 Pep Ole e 1.3 63-99GLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWA 81 Pep Ole e 1.4  86-120 GRKDCNEIPTEGWAKPSLKFKLNTVNGTTRTVNPL 82 Pep Ole e 1.5 107-145 LNTVNGTTRTVNPLGFFKKEALPKCAQVYNKLGMYPP 83 NM Pep Par j 2.1 31-60GEEACGKVVQDIMPCLHFVKGEEKEPSKEC 84 Pep Par j 2.2 45-80CLHFVKGEEKEPSKECCSGTKKLSEEVKTTEQKREA 85 Pep Par j 2.3 60-96CCSGTKKLSEEVKTTEQKREACKCIVRATKGISGIKN 86 Pep Par j 2.4  97-133ELVAEVPKKCDIKTTLPPITADFDCSKIQSTIFRGYY 87 Der p 1 Pep 1  1-30TNACSINGNAPAEIDLRQMRTVTPIRMQGG 88 Der p 1 Pep 2 52-84NQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQ 89 Der p 1 Pep 3  85-115HNGVVQESYYRYVAREQSCRRPNAQRFGISN 90 Der p 1 Pep 4  99-135REQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTH 91 Der p 1 Pep 5 145-175KDLDAFRHYDGRTIIQRDNGYQPNYHAVNIV 92 Der p 1 Pep 6 155-187GRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWI 93 Der p 1 Pep 7 175-208VGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI 94 Der p 1 Pep 8 188-222VRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVIL 95 Der p 1 Pep 1.2  1-41TNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFS 143 GVA Der p 1 Pep 2.2 42-82ATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRG 144 IEYIQ Der p 1 Pep 9 27-57MQGGCGSCWAFSGVAATESAYLAYRNQSLD 145 Der p 2 Pep 1  1-33DQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGK 96 Der p 2 Pep 2 21-51CHGSEPCIIHRGKPFQLEAVFEANQNSKTAK 97 Der p 2 Pep 3 42-73EANQNSKTAKIEIKASIEGLEVDVPGIDPNAC 98 Der p 2 Pep 4  62-103EVDVPGIDPNACHYMKCPLVKGQQYDIKYTWIVPKIAP 99 KSEN Der p 2 Pep 5  98-129APKSENVVVTVKVMGDNGVLACAIATHAKIRD 100 Der p 5 Pep 1  1-35MEDKKHDYQNEFDFLLMERIHEQIKKGELALFYLQ 101 Der p 5 Pep 2 25-60KKGELALFYLQEQINHFEEKPTKEMKDKIVAEMDTI 102 Der p 5 Pep 3 65-95DGVRGVLDRLMQRKDLDIFEQYNLEMAKKSG 103 Der p 5 Pep 4  78-114DLDIFEQYNLEMAKKSGDILERDLKKEEARVKKIEV 104 Der p 7 Pep 1  1-30DPIHYDKITEEINKAVDEAVAAIEKSETFD 105 Der p 7 Pep 2 20-50VAAIEKSETFDPMKVPDHSDKFERHIGIIDL 106 Der p 7 Pep 3 50-80LKGELDMRNIQVRGLKQMKRVGDANVKSEDG 107 Der p 7 Pep 4  90-125VHDDVVSMEYDLAYKLGDLHPNTHVISDIQDFVVEL 108 Der p 7 Pep 5 125-155LSLEVSEEGNMTLTSFEVRQFANVVNHIGGL 109 Der p 7 Pep 6 165-198LSDVLTAIFQDTVRAEMTKVLAPAFKKELERNNQ 110 Der p 10 Pep 1  1-35MEAIKKKMQAMKLEKDNAIDRAEIAEQKARDANLR 111 Der p 10 Pep 2 36-70AEKSEEEVRALQKKIQQIENELDQVQEQLSAANTK 112 Der p 10 Pep 3  71-110LEEKEKALQTAEGDVAALNRRIQLIEEDLERSEERLKIA 113 T Der p 10 Pep 4 111-145AKLEEASQSADESERMRKMLEHRSITDEERMEGLE 114 Der p 10 Pep 5 140-170RMEGLENQLKEARMMAEDADRKYDEVARKLA 115 Der p 10 Pep 6 175-205DLERAEERAETGESKIVELEEELRVVGNNLK 116 Der p 10 Pep 7 210-250SEEKAQQREEAHEQQIRIMTTKLKEAEARAEFAERSVQ 117 KLQ Der p 10 Pep 8 250-284QKEVDRLEDELVHEKEKYKSISDELDQTFAELTGY 118 Der p 21 Pep 1  1-35MFIVGDKKEDEWRMAFDRLMMEELETKIDQVEKGL 119 Der p 21 Pep 2 35-72LHLSEQYKELEKTKSKELKEQILRELTIGENFMKGAL 120 Der p 21 Pep 3  70-100GALKFFEMEAKRTDLNMFERYNYEFALESIK 121 Der p 21 Pep 4  90-122YNYEFALESIKLLIKKLDELAKKVKAVNPDEYY 122 Der p 23 Pep 1  1-32MANDNDDDPTTTVHPTTTEQPDDKFECPSRFG 123 Der p 23 Pep 2 15-48PTTTEQPDDKFECPSRFGYFADPKDPHKFYICSN 124 Der p 23 Pep 3 32-70GYFADPKDPHKFYICSNWEAVHKDCPGNTRWNEDEE 125 TCT Der p 23 Pep 4 32-60GYFADPKDPHKFYICSNWEAVHKDCPGNT 146 Der p 23 Pep 5 42-70KFYICSNWEAVHKDCPGNTRWNEDEETCT 147 Der p 23 Pep 6  32-70*GYFADPKDPHKFYISSNWEAVHKDSPGNTRWNEDEETS 148 (Cys T ->Ser) Bet v 1 Pep 130-59 LFPKVAPQAISSVENIEGNGGPGTIKKISF 126 Bet v 1 Pep 2 50-79GPGTIKKISFPEGFPFKYVKDRVDEVDHTN 127 Bet v 1 Pep 3  75-104VDHTNFKYNYSVIEGGPIGDTLEKISNEIK 128 Bet v 1 Pep A 30-74LFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVK 143 DRVDE Bet v 1 Pep B  60-104PEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEK 144 ISNEIKI Fel d 1 chain 1 1-34 EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVC 129 Pep 1 Fel d 1 chain 135-70 LENARILKNCVDAKMTEEDKENALSLLDKIYTSPLC 130 Pep 2 Fel d 1 chain 2 1-34 VKMAITCPIFYDVFFAVANGNELLLDLSLTKVNAC 131 Pep 1 Fel d 1 chain 235-63 TEPERTAMKKIQDCYVENGLISRVLDGLVC 132 Pep 2 Fel d 1 chain 2 64-92CMTTISSSKDCMGEAVQNTVEDLKLNTLGR 133 Pep 3 Ph1 p 5 Pep 1  98-128CGAASNKAFAEGLSGEPKGAAESSSKAALTSK 134 Ph1 p 5 Pep 2 26-58ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKC 135 Ph1 p 5 Pep 3 132-162AYKLAYKTAEGATPEAKYDAYVATLSEALRIC 136 Ph1 p 5 Pep 4 217-246CEAAFNDAIKASTGGAYESYKFIPALEAAVK 137 Phl p 5 Pep 5 252-283TVATAPEVKYTVFETALKKAITAMSEAQKAAKC 138 Ph1 p 5 Pep 6 176-212CAEEVKVIPAGELQVIEKVDAAFKVAATAANAAPAND 139 K Ph1 p 5 Pep 1a  93-128CFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK 141 Ph1 p 5 Pep 2b 26-53ADLGYGPATPAAPAAGYTPATPAAPAEAC 142 Ph1 p 5 Pep 7 59-91ATTEEQKLIEKINAGFKAALAAAAGVQPADKYR 22 Ph1 p 1 Pep 1 151-171HVEKGSNPNYLALLVKYVNGDGDVVAVC 1 Ph1 p 1 Pep 2  87-117EPVVVHITDDNEEPIAPYHFDLSGHAFGAMAC 2 Ph1 p 1 Pep 3  1-30IPKVPPGPNITATYGDKWLDAKSTWYGKPTGC 3 Ph1 p 1 Pep 4 43-70GYKDVDKPPFSGMTGCGNTPIFKSGRGC 4 Ph1 p 1 Pep 5 212-241CVRYTTEGGTKTEAEDVIPEGWKADTSYESK 5 Ph1 p 2 Pep 1  1-33VPKVTFTVEKGSNEKHLAVLVKYEGDTMAEVELC 6 Ph1 p 2 Pep 2 28-39CVEKGSNEKHLAVLVKYEGDTMAEVELREHGSD 7 Ph1 p 2 Pep 3 34-65REHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNC 8 Ph1 p 2 Pep 4 66-96CFRFLTEKGMKNVFDDVVPEKYTIGATYAPEE 9 Ph1 p 6 Pep 1 23-54GKATTEEQKLIEDVNASFRAAMATTANVPPAD 10 Ph1 p 6 Pep 2 56-90YKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYN 11 Ph1 p 6 Pep 3  95-127AADHAAPEDKYEAFVLHFSEALRIIAGTPEVHA 12 Ph1 p 6 Pep 4  73-114DAVSKAPQLVPKLDEVYNAAYNAADHAAPEDKY 13 *) Cysteins exchanged with serins(marked in bold)

The terms “fragments thereof” and “sequence variations thereof” refer topeptides which are deduced from the allergen-derived molecules disclosedherein and show biochemical properties (e.g. the capacity to prevent IgEbinding to the allergen from which those molecules are derived from)which are comparable or identical to said allergen-derived molecules.The fragments of the present invention comprise at least 5, preferablyat least 7, more preferably at least 10, successive and/or a maximum of95%, preferably a maximum of 90%, more preferably a maximum of 80% aminoacid residues of the allergen-derived molecule. The term “sequencevariation” includes modifications of the peptides such as fragmentation(see above), amino acid substitutions (in particular cysteine ormethionine residues may be exchanged with serine, alanine or othernatural or non-natural amino acids or amino acid derivatives), deletionsor additions. “Sequence variation” refers also to said allergen-derivedmolecules of the above table, wherein at least 1, preferably at least 2,more preferably at least 3, even more preferably at least 4 (5, 6, 7, 8,9, 10, 15, 20) amino acid residues are added to the C- and/orN-terminus.

It is noted that the allergen referred to herein as “clone 30 allergen”is an allergen derived from the house dust mite Dermatophagoidespteronyssinus and consists of the following sequence:MANDNDDDPTTTVHPTTTEQPDDKFECPSRFGYFADPKDPHKFYICSNWEAVHKDCP GNTRWNEDEETCT(SEQ ID No. 140; see also WO 2007/124524). In the meantime, the allergenname Der p 23 has been assigned to clone 30 allergen. This means thatDer p 23 and clone 30 allergen are synonyms.

According to the present invention also peptides are encompassed whichare at least 80% identical, preferably 90% identical, to the aminosequences disclosed above.

According to a preferred embodiment of the present invention the surfacepolypeptide of the virus of the hepadnaviridae family or at least onefragment thereof comprises at least two B-cell binding peptide fragmentsderived from at least one wild-type allergen fused to its N-terminus andat least two B-cell binding peptide fragments derived from at least onewild-type allergen fused to its C-terminus.

In a particularly preferred embodiment of the present invention at leasttwo of said at least three B-cell binding peptides are identical.

The polypeptide of the present invention can be used as vaccine in thetreatment or prevention of an allergy in a human or animal.

The polypeptide is preferably administered to an individual in theamount of 0.01 microgram per kg body weight to 5 mg/kg body weight,pref-erably 0.1 microgram per kg body weight to 10 microgram per kg bodyweight.

According to a particularly preferred embodiment of the presentinvention the polypeptides of the present invention are administered toan individual in an amount of at least 10 μg, preferably at least 20 μg,per polypeptide. The maximum amount of polypeptides to be administeredcan be varied but is preferably below 100 μg, more preferably below 50μg, even more preferably 40 μg or less, per polypeptide.

The amount of polypeptides that may be combined with excipients toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. The dose of the vaccine mayvary according to factors such as the disease state, age, sex and weightof the individual, and the ability of antibody to elicit a desiredresponse in the individual. Dosage regime may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. The dose ofthe vaccine may also be varied to provide optimum preventative doseresponse depending upon the circumstances. For instance, thepolypeptides and vaccine of the present invention may be administered toan individual at intervals of several days, one or two weeks or evenmonths depending always on the level of allergen-specific IgG induction.

In a preferred embodiment of the present invention thepolypeptide/vaccine is applied between 2 and 10, preferably between 2and 7, even more preferably up to 5 and most preferably up to 3 times.In a particularly preferred embodiment the time interval between thesubsequent vaccinations is chosen to be between 2 weeks and 5 years,preferably between 1 month and up to 3 years, more preferably between 2months and 1.5 years. The repeated administration of the peptide/vaccineof the present invention may maximize the final effect of a therapeuticvaccination.

According to a particularly preferred embodiment of the presentinvention three or more B-cell binding peptides selected from the groupconsisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 9,SEQ ID No. 137, SEQ ID No. 139, SEQ ID No. 142 and SEQ ID No. 10 arebound N- and C-terminally to a surface polypeptide of the virus of thehepadnaviridae family, preferably the hepatitis PreS polypeptide orfragments thereof.

The polypeptides of the present invention comprising the at least threeB-cell binding peptides derived from at least one wild-type allergen arepreferably selected from the group consisting of SEQ ID No. 14, SEQ IDNo. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and SEQ ID No. 19.

Another aspect of the present invention relates to a nucleic acidmolecule encoding a polypeptide according to the present invention.

Another aspect of the present invention relates to a vector comprising anucleic acid molecule according to the present invention.

Said vector is preferably an expression vector.

The vector harbouring the nucleic acid molecule of the present inventionmay be used for cloning purposes or for the production of expressionvectors. Said vector can be a plasmid, cosmid, virus, bacteriophage orany other vector commonly used in genetic engineering, and can include,in addition to the nucleic acid molecule of the invention, eukaryotic orprokaryotic elements for the control of the expression, such asregulatory sequences for the initiation and the termination of thetranscription and/or translation, enhancers, promoters, signal sequencesand the like.

According to a preferred embodiment of the present invention the vectoris a bacterial, fungal, insect, viral or mammalian vector.

The vector of the present invention may preferably be employed forcloning and expression purposes in various hosts like bacteria, yeasts,filamentous fungi, mammalian cells, insect cells, plant cells or anyother prokaryotic or eukaryotic cells. Therefore, said vector comprisesbesides a nucleic acid encoding for a hypoallergenic molecule or fusionprotein according to the present invention host specific regulatorysequences.

Another aspect of the present invention relates to a host comprising anucleic acid molecule or a vector according to the present invention.

The nucleic acid molecule and the vector according to the presentinvention may be introduced into a suitable host. Said molecule may beincorporated into the genome of the host. The vector may existextrachromosomally in the cytoplasm or incorporated into the chromosomeof the host.

Yet another aspect of the present invention relates to an antibodydirected against a hypoallergenic molecule, hypoallergenic fusionprotein or a fusion protein according to the present invention.

Another aspect of the present invention relates to a vaccine formulationcomprising at least one, preferably at least two, more preferably atleast three, even more preferably at least 4, polypeptide according tothe present invention.

In a particularly preferred embodiment of the present invention thevaccine comprises at least one, preferably at least two, preferably atleast three, preferably at least four, preferably at least 5,polypeptides having an amino acid sequence selected from the groupconsisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No.17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ IDNo. 149, SEQ IDNo. 150, SEQ ID No. 151 and SEQ ID No. 152.

Depending on the composition such a vaccine can be used in the treatmentand/or prevention of grass pollen allergies, birch pollen allergies,house dust mite allergies or a combination of those allergies inindividuals suffering from such allergies or being at risk to suffertherefrom.

The term “preventing”, as used herein, covers measures not only toprevent the occurrence of disease, such as risk factor reduction, butalso to arrest its progress and reduce its consequences onceestablished. “Preventing” means also to prevent sensitization of anindividual being at risk to get an allergy.

As used herein, the term “treatment” or grammatical equivalentsencompasses the improvement and/or reversal of the symptoms of disease(e.g., allergy). A compound which causes an improvement in any parameterassociated with disease when used in the screening methods of theinstant invention may thereby be identified as a therapeutic compound.The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures.

According to one of the most preferred embodiment of the presentinvention the vaccine comprises polypeptides having amino acid sequenceSEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17.

According to another preferred embodiment of the present invention thevaccine comprises polypeptides having amino acid sequence SEQ ID No. 18and/or SEQ ID No. 19.

According to a particularly preferred embodiment of the presentinvention the vaccine comprises polypeptides of the present inventionwhich comprise allergen fragments derived from house dust miteallergens. Particularly preferred are amino acid residues 1 to 33, 21 to51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acid residues 1to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p7, amino acid residues 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Derp 21, amino acids 1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32to 70 (Cys->Ser) of Der p 23, amino acid residues 1 to 30, 1 to 41, 27to 57, 42 to 82, 52 to 84, 85 to 115, 99 to 135, 145 to 175, 155 to 187,175 to 208 or 188 to 222 of Der p 1. Most preferably, the vaccinecomprises at least one of the polypeptides SEQ ID No. 149 to 152 (shownin FIGS. 18 A-18 D).

In a particularly preferred embodiment the polypeptide/vaccine of thepresent invention is administered 4 times per treatment year over atotal treatment period of 1 to 5 years, preferably over 2 to 3 years. Ofsaid 4 yearly administrations 3 are applied within a period of 6 to 12,preferably 8, weeks having intervals of 3 to 6 weeks, preferably 4weeks, between administration 1 and 2 and another 3 to 6 weeks,preferably 4 weeks, between administration 2 and 3. The fourthadministration is applied 3 to 7 months after the third administration.If the total treatment period exceeds 1 year, the same dosing regimen isapplied in the following treatment years.

For the treatment of seasonal allergies (e.g. pollen allergies such asgrass pollen allergy or birch pollen allergy) administration 1, 2, and 3are preferably scheduled before the respective season with allergenexposure (pollen season), and the fourth administration is scheduledafter the season.

The vaccine formulation according to the present invention may beformulated as known in the art and necessarily adapted to the way ofadministration of said vaccine formulation.

Preferred ways of administration of the vaccine formulation (of thepresent invention) include all standard administration regimes describedand suggested for vaccination in general and allergy immunotherapyspecifically (orally, transdermally, intraveneously, intranasally, viamucosa, rectally, etc). However, it is particularly preferred toadminister the molecules and proteins according to the present inventionsubcutaneously or intramusculary.

The vaccine formulation according to the present invention may onlycomprise a viral capsid protein or fragments thereof of a member of thegenus of hepadnaviridae.

Said formulation preferably further comprises at least one adjuvant,pharmaceutical acceptable excipient and/or preservative.

In order to increase the immunogenicity of the hypoallergenic moleculesaccording to the present invention, adjuvants, for instance, may be usedin a medicament according to the present invention. An adjuvantaccording to the present invention is an auxiliary agent which, whenadministered together or in parallel with an antigen, increases itsimmunogenicity and/or influences the quality of the immune response.Hence, the adjuvant can, e.g., considerably influence the extent of thehumoral or cellular immune response. Customary adjuvants are, e.g.,aluminum compounds, lipid-containing compounds or inactivatedmycobacteria.

Generally, adjuvants can be of different forms, provided that they aresuitable for administration to human beings. Further examples of suchadjuvants are oil emulsions of mineral or vegetal origin, mineralcompounds such as aluminium phosphate or hydroxide, or calciumphosphate, bacterial products and derivatives, such as P40 (derived fromthe cell wall of Corynebacterium granulosum), monophosphoryl lipid A(MPL, derivative of LPS) and muramyl peptide derivatives and conjugatesthereof (derivatives from mycobacterium components), alum, incompleteFreund's adjuvant, liposyn, saponin, squalene, etc. (see, e.g., Gupta R.K. et al. (Vaccine 11:293-306 (1993)) and Johnson A. G. (Clin.Microbiol. Rev. 7:277-289). The medicament of the present inventioncomprises most preferably alum as adjuvant.

Another preferred embodiment of the present invention is a combinationof more than one fusion protein containing hypoallergenic peptides andthe hepatitis B pre S protein. These combinations may be derived frompeptides from a single allergen or from different allergens of the sameallergen source or from several different allergen source.

A preferred embodiment of the present invention relates to a mixture offour fusion proteins containing hypoallergenic peptides from Phl p 1,Phl p 2, Phl p 5, and Phl p 6 and the hepatitis B virus preS protein.

Another preferred embodiment of the present invention relates to afusion protein or a mixture of 2 fusion proteins containinghypoallergenic peptides from Bet v 1 and the hepatitis B virus PreSprotein.

Yet another preferred embodiment of the present invention relates to amixture of at least 2 fusion proteins containing hypoallergenic peptidesfrom house dust mite allergens, most preferably selected from Der p 1,Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 and the hepatitis Bvirus PreS protein. Most preferably, the mixture contains 3 fusionproteins containing hypoallergenic peptides derived from Der p 1, Der p2, and Der p 23. It is particularly preferred that the mixture comprisesat least one, preferably at least two, more preferably at least three,of the polypeptides shown in SEQ ID No. 149 to 152 (see also FIGS. 18A-18 D).

Generally, specific vaccine formulations according to the presentinvention can be prepared for the treatment or prevention of differentallergies by combination of hypoallergenic polypeptides of the inventionrepresenting the clinically relevant allergens of an allergen source.Methods to determine the clinically relevant allergens of an allergensource are known in the art and have been described before (Valenta andNiederberger, 2007, J Allergy Clin Immunol, 119 (4): 826-830). In apreferred embodiment, the hypoallergenic polypeptides of said specificvaccine formulation are adsorbed to an adjuvant which can be used inhuman (e.g. aluminium hydroxide), and the mixture is administered 3-4times per year for 1-3 years applying more than 10 μg of eachpolypeptide present in the vaccine formulation per dose.

According to another preferred embodiment of the present invention saidformulations comprise 10 ng to 1 g, preferably 100 ng to 10 mg,especially 0.5 μg to 200 μg of said hypoallergenic molecule or antibody.

Another aspect of the present invention relates to the use of ahypoallergenic protein or an antibody according to the present inventionfor manufacturing a medicament for the treatment or prevention of aviral infection and/or an allergy in a human or animal.

Said medicament preferably further comprises at least one adjuvant,pharmaceutical acceptable excipient and/or preservative.

The medicament according to the present invention may be used for active(administration of the hypoallergenic protein and/or molecules of theinvention) as well as for passive immunization (antibodies directed tothe hypoallergenic protein and/or molecules of the invention).

According to a preferred embodiment of the present invention saidmedicament comprises 10 ng to 1 g, preferably 100 ng to 10 mg,especially 0.5 μg to 200 μg of said hypoallergenic molecule, nucleicacid molecule, vector, host or antibody.

The medicament is preferably administered to an individual in amount of0.01 μg/kg body weight to 5 mg/kg body weight, preferably 0.1 μg/kg bodyweight to 10 μg/kg body weight.

In a particularly preferred embodiment, the medicament is administeredin a dose containing an absolute amount of 5-200 μg, more preferably10-80 μg, most preferably 20-40 μg of each included hypoallergenicpolypeptide

The particular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory. Empirical considerations, such as the half life, will generallycontribute to determination of the dosage. Frequency of administrationmay be determined and adjusted over the course of therapy.

Most preferably, the dosing regimen for the medicament will consist of 4yearly subcutaneous injections of one and the same dose over a totaltreatment period of 2 to 3 years. Of said 4 yearly subcutaneousinjections 3 are applied within a period of 6 to 12, preferably 8, weekshaving intervals of 3 to 6 weeks, preferably 4 weeks, between injection1 and 2 and another 3 to 6 weeks, preferably 4 weeks, between injection2 and 3. The fourth injection is applied 4 to 6 months after the thirdadministration. The same dosing regimen is applied in the followingtreatment years.

For the treatment of seasonal allergies (e.g. pollen allergies such asgrass pollen allergy or birch pollen allergy) administration 1, 2, and 3are preferably scheduled before the respective season with allergenexposure (pollen season), and the fourth administration is scheduledafter the season.

The individual to whom the medicament according to the present inventionis administered is preferably an individual or animal which is having oris at risk of having an allergy.

Subjects having or at risk of having an allergy, allergic condition,allergic disorder or allergic disease include subjects with an existingallergic condition or a known or a suspected predisposition towardsdeveloping a symptom associated with or caused by an allergic condition.Thus, the subject can have an active chronic allergic condition,disorder or disease, an acute allergic episode, or a latent allergiccondition, disorder or disease. Certain allergic conditions areassociated with seasonal or geographical environmental factors. Thus, atrisk subjects include those at risk from suffering from a conditionbased upon a prior personal or family history, and the season orphysical location, but which the condition or a symptom associated withthe condition may not presently manifest itself in the subject.

The administration of the medicament according to the present invention,which comprises at least one hypoallergenic molecule as describedherein, to an individual may prevent sensitization of said individual ormay induce an appropriate immune response to allergens. If themedicament of the present invention is used to prevent sensitization, itshould be administered to an individual prior to the first contact withsaid allergen. Therefore, it is preferred to administer the medicamentaccording to the present invention to neonates and children. It turnedout that also the administration of the medicament according to thepresent invention to pregnant individuals will induce the formation ofantibodies directed against allergens in the unborn child. It isespecially beneficiary to use hypoallergenic molecules according to thepresent invention for such therapies, because due to the lack ofallergen-specific T-cell epitopes side effects occurring in the courseof allergen immunotherapy can significantly be reduced or even becompletely avoided.

Another aspect of the present invention relates to the use of a viralcapsid protein from a virus of the family of hepadnaviridae as a carrierin medicaments or vaccines.

One of the advantages of such a carrier is that not only the antigenfused or conjugated thereon may be exposed to the immune system, butalso an immune response against the capsid protein of a hepadnavirus isinduced. Consequently, such a vaccination may lead to the preventionand/or treatment of diseases caused by hepadnaviruses. The virus ispreferably of the species of human hepatitis B virus.

Another aspect of the present invention relates to a hypoallergenicmolecule derived from Phl p 5 (Genbank Nr. X7435) having a C- and/orN-terminal truncation and lacking substantially IgE-binding capacity.

Grass pollen is one of most potent outdoor seasonal sources of airborneallergens responsible for hay fever and allergic asthma.

More than 40% of allergic individuals display IgE-reactivity with grasspollen allergens, which are divided into more than 11 groups. More than80% of the grass pollen allergic patients react with group 5 allergens.

Group 5 allergens are non-glycosylated, highly homologous proteins witha molecular mass range from 25-33kD. Several group 5 allergens have beencloned and/or immunologically characterized.

The trial to reduce the allergenic activity by introducingpointmutations, mutations of several amino acids in row or deletionsshowed no effect (Schramm G, et al. J Immunol 1999; 162: 2406-1435).IgE-binding regions of Phl p 5 (Flicker S, et al. J Immunol 2000; 165:3849-3859) have already been described and the three-dimensionalstructure has been solved (Maglio O, et al. 2002. Protein Eng.15:635-642).

It turned out that in particular the Phl p 5 peptides according to thepresent invention, which are C- and/or N-terminally truncated and lackIgE-binding capacity, may be employed for the active vaccination ofindividuals.

According to a preferred embodiment of the present invention thetruncated molecule substancially lacks T-cell epitopes and, thus lacksPhl p 5-specific T-cell reactivity.

As already outlined above, late side effects of allergen immunotherapycan be significantly reduced or even be avoided if the hypoallergenicmolecules substantially lack allergen-specific T-cell epitopes.

Truncated Phl p 5 molecules lacking T-cell epitopes are composed ofamino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58 or 252 to 283 ofPhl p 5 or fragments or sequence variations thereof.

In particular these truncated molecules substantially show low or noallergen-specific T-cell reactivity and are, nevertheless, able toprovoke an appropriate immune response directed against the wild-typeallergen.

According to another preferred embodiment of the present invention thehypoallergenic truncated Phl p 5 is composed of amino acids 132 to 162,217 to 246 or 176 to 212 of Phl p 5 or sequence variations thereof.

These hypoallergenic molecules comprise one or more T-cell epitopes butlack IgE-binding capacity.

Another preferred embodiment of the present invention are truncated Phlp 1 molecules lacking T-cell epitopes, which are composed of amino acids1 to 30, 43 to 70, 87 to 117, 151 to 171 or 214 to 241 of Phl p 1 orsequence variations thereof fused to a viral carrier protein, preferablethe Hep B pre S protein.

Another preferred embodiment of the present invention are truncated Phlp 2 molecules lacking T-cell epitopes, which are composed of amino acids1 to 33, 8 to 39, 34 to 65 or 66 to 96 of Phl p 2 or sequence variationsthereof fused to a viral carrier protein, preferrably the Hep B pre Sprotein.

Another preferred embodiment of the present invention are truncated Phlp 6 molecules lacking T-cell epitopes, which are composed of amino acids23 to 54, 56 to 90, 73 to 114 or 95 to 127 of Phl p 6 or sequencevariants thereof fused to a viral carrier protein, preferrably the Hep Bpre S protein.

Another preferred embodiment of the present invention refers totruncated Bet v 1 molecules lacking T-cell epitopes, which are composedof amino acids 30 to 59, 50 to 79, 75 to 104, 30 to 74 or 60 to 104 ofBet v 1.

Another preferred embodiment of the present invention are combinationsor mixtures of truncated Phleum pratense molecules lacking T-cellepitopes, fused to a viral carrier protein, preferrably the Hep B pre Sprotein, as described above.

A preferred embodiment of the present invention are combinations ormixtures of truncated Phleum pratense molecules lacking T-cell epitopes,which are composed of one each such fusion protein from truncated Phl p1, Phl p 2, Phl p 5, and Phl p 6, as described above.

Another aspect of the present invention relates to a hypoallergenicmolecule derived from Fel d 1 (Genbank Nr. X62477 and X62478) having aC- and/or N-terminal truncation and lacking IgE-binding capacity.

Allergies to animals affect up to 40% of allergic patients. In thedomestic environment, allergies to the most popular pets, cats and dogs,are particularly prevalent and connected with perennial symptoms. Animalallergens are present in dander, epithelium, saliva, serum or urine.Exposure to the allergens can occur either by direct skin contact or byinhalation of particles carrying the allergens. The major cat and dogallergens were shown to be present widespread and could even be detectedin non-pet owning households and in public places, e.g., schools. Thiscan be attributed to the high and increasing number of householdskeeping pets in industrialized countries (about 50%) and the highstability of the allergens, which are carried off and distributed.

Fel d 1 was identified as the major cat allergen, which is recognized bymore than 90% of cat allergic patients. Fel d 1 represents a 38 kDaacidic glycoprotein of unknown biological function. It consists of twoidentical non-covalently linked heterodimers, which, again, are composedof two polypeptide chains antiparallely linked by three disulfide bonds.Chain 1 and chain 2 are encoded on different genes, each consisting of 3exons. Recombinant Fel d 1 (rFel d 1), expressed as a chain 2- to chain1 fusion protein, has been generated in E. coli. This recombinant Fel d1 is able to completely mimic the immunological properties of thewild-type allergen.

Peptides derived from the major cat allergen Fel d 1, and lackingIgE-binding capacity are suitable, e.g., for immunotherapy andprophylactic allergy vaccination. The Fel d 1-derived syntheticpeptides—like the Phl p 5 and allergen-derived peptides disclosedherein—are capable of inducing an IgG response, i.e., the production ofso called “blocking antibodies” or “protective antibodies”. Theseantibodies prevent IgE-binding to the allergen Fel d 1. A significantreduction in allergic symptoms may thus be achieved.

According to a preferred embodiment of the present invention thetruncated molecule exhibits reduced T-cell reactivity.

In order to avoid or to significantly reduce late side effects the Fel d1 derived hypoallergenic molecule exhibits reduced T-cell reactivity asdefined in the present invention.

The truncated Fel d 1 is preferably composed of amino acids 1 to 34 or35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to92 of chain 2 of Fel d 1 or sequence variations thereof.

Another aspect of the present invention relates to hypoallergenicmolecules being composed of or comprising amino acids 1 to 33, 21 to 51,42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, aminoacids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids1 to 32, 15 to 48 or 32 to 70, 32 to 60, 52 to 84, 32 to 70 (Cys->Ser)of Der p 23, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 ofAlt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 ofOle e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200,225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80,81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variationsthereof.

Methods for the production of fusion proteins are well known in the artand can be found in standard molecular biology references such asSambrook et al. (Molecular Cloning, 2nd ed., Cold Spring HarborLaboratory Press, 1989) and Ausubel et al. (Short Protocols in MolecularBiology, 3rd ed; Wiley and Sons, 1995). In general, a fusion protein isproduced by first constructing a fusion gene which is inserted into asuitable expression vector, which is, in turn, used to transfect asuitable hosT-cell. In general, recombinant fusion constructs areproduced by a series of restriction enzyme digestions and ligationreactions which result in the desired sequences being incorporated intoa plasmid. If suitable restriction sites are not available, syntheticoligonucleotide adapters or linkers can be used as is known by thoseskilled in the art and described in the references cited above. Thepolynucleotide sequences encoding allergens and native proteins can beassembled prior to insertion into a suitable vector or the sequenceencoding the allergen can be inserted adjacent to a sequence encoding anative sequence already present in a vector. Insertion of the sequencewithin the vector should be in frame so that the sequence can betranscribed into a protein. It will be apparent to those of ordinaryskill in the art that the precise restriction enzymes, linkers and/oradaptors required as well as the precise reaction conditions will varywith the sequences and cloning vectors used. The assembly of DNAconstructs, however, is routine in the art and can be readilyaccomplished by a person skilled in the art.

It is a specific and unexpected advantage, that the fusion proteinsderived from truncated hypoallergenic allergen molecules and the humanhepatitis B pre S protein can be reproducibly expressed in standardexpression systems and easily be manufactured produced in high yieldwith processes and reproducibly in standard expression systems known toa person skilled in the art, most particularly by using in anEscherichia coli as expression system. Such manufacturing processtypically comprise the expression of the molecules according to theinvention by the cultivation of cells in a bioreactor (e.g. in afermenter, shake flask), followed by cell harvest (e.g. by filtration,centrifugation, etc.) and cell disruption (e.g. by high-pressurehomogenization, sonication, freeze/thaw cycles, enzymatic or chemicalcell lysis, etc.), purification of the molecules (e.g. bychromatography, filtration, precipitation, ultra/diafiltration, etc.)and final product formulation. In order to obtain a high yield of themolecules according to the invention, preferably high-cell densitycultivation processes are employed, by application of fed-batchfermentation.

Another aspect of the present invention relates to a nucleic acidmolecule coding for a hypoallergenic molecule and a fusion proteinaccording to the present invention.

The nucleic acid molecule of the present invention may be employed,e.g., for producing said molecules recombinantly.

Said nucleic acid molecule may—according to another aspect of thepresent invention—be comprised in a vector.

This vector is preferably an expression vector.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The present invention is further illustrated by the following figuresand examples, however, without being restricted thereto.

FIG. 1 A shows a schematic overview of vector HBV_Phlp1_(—)4xP5

FIG. 1 B shows a schematic overview of vector HBV_Phlp2_(—)4xP3

FIG. 1 C shows a schematic overview of vector HBV_Phlp5_V2

FIG. 1 D shows a schematic overview of vector HBV_Phlp6_(—)4xP1

FIG. 2 A shows the primary sequence of fusion protein HBV_Ph1P1_(—)4xP5(BM321, sequence ID Nr. 14)

FIG. 2 B shows the primary sequence of fusion protein HBV_Phlp2_(—)4xP3(BM322, sequence ID Nr. 15)

FIG. 2 C shows the primary sequence of fusion protein HBV_Phlp5_V2(BM325, sequence ID Nr. 16)

FIG. 2 D shows the primary sequence of fusion protein HBV_Phlp6_(—)4xP1(B326, sequence ID Nr. 17)

FIG. 2 E shows the primary sequence of fusion protein HBV_Betv1_(—)4 PA(BM31a, sequence ID Nr. 18)

FIG. 2 F shows the primary sequence of fusion protein HBV_Betv1_(—)2PA2PB (BM31, sequence ID Nr. 19)

FIG. 2 G shows the primary sequence of fusion protein HBV_Phlp5_V1(sequence ID No. 20)

FIG. 3 A shows a Coomassie Blue stained 12% SDS Page gel containingpurified fusion protein HBV_Phlp1_(—)4xP5 (BM 321, lane 1 and 10:5 ugmolecular marker, lane 2, 3, 11 and 12 5 ug BM321, lane 4 and 13 2 ugBM321, lane 5 and 14 1 ug BM321, lane 6 and 15 0.5 ug BM321, lane 7 and16 0.25 ug BM321, lane 8 and 17 0.1 ug BM 321, lane 9 and 18 0.05 ugBM321). Lanes 1 to 9 are under reducing and lanes 10-18 undernon-reducing conditions.

FIG. 3 B shows a Coomassie Blue stained 12% SDS Page gel containingpurified fusion protein HBV_Phlp2_(—)4xP3 (BM 322, lane 1 and 10:5 ugmolecular marker, lane 2, 3, 11 and 12 5 ug BM322, lane 4 and 13 2 ugBM322, lane 5 and 14 1 ug BM322, lane 6 and 15 0.5 ug BM322, lane 7 and16 0.25 ug BM322, lane 8 and 17 0.1 ug BM 322, lane 9 and 18 0.05 ugBM322). Lanes 1 to 9 are under reducing and lanes 10-18 undernon-reducing conditions.

FIG. 3 C shows a Coomassie Blue stained 12% SDS Page gel containingpurified fusion protein HBV_Phlp5_V2 (BM 325, lane 1 and 10:5 ugmolecular marker, lane 2, 3, 11 and 12 5 ug BM325, lane 4 and 13 2 ugBM325, lane 5 and 14 1 ug BM325, lane 6 and 15 0.5 ug BM325, lane 7 and16 0.25 ug BM325, lane 8 and 17 0.1 ug BM 325, lane 9 and 18 0.05 ugBM325). Lanes 1 to 9 are under reducing and lanes 10-18 undernon-reducing conditions.

FIG. 3 D shows a Coomassie Blue stained 12% SDS Page gel containingpurified fusion protein HBV_Phlp6_(—)4xP1 (BM 326, lane 1 and 10:5 ugmolecular marker, lane 2, 3, 11 and 12 5 ug BM326, lane 4 and 13 2 ugBM326, lane 5 and 14 1 ug BM326, lane 6 and 15 0.5 ug BM326, lane 7 and16 0.25 ug BM326, lane 8 and 17 0.1 ug BM 326, lane 9 and 18 0.05 ugBM326). Lanes 1 to 9 are under reducing and lanes 10-18 undernon-reducing conditions.

FIGS. 4 A-4 B demonstrate the lack of IgE reactivity of fusion peptidesderived from grass pollen allergens. IgE binding of fusion proteins incomparison to the complete allergen was tested by IgE dot-blot assay.Sera from the indicated number of grass pollen allergic patients wereincubated with dotted proteins and bound IgE was detected with125I-labelled anti-human IgE. No IgE binding was detected for any of thefour peptide-carrier fusion proteins.

FIG. 4 A shows the results from the dot blot assay usingHBV_Phlp1_(—)4XP5 (BM321);

FIG. 4 B shows the results from the dot blot assay usingHBV_Phlp2_(—)4xP3 (BM322);

FIG. 4 C shows the results from the blot assay using HBV_Phlp5_V2(BM325);

FIG. 4 D shows the results from form the dot blot assay usingHBV_Phlp6_(—)4xP1 (BM326).

FIG. 5 shows the low allergenic activity of grass pollen allergenderived fusion protein HBV_Phlp1_(—)4xP5 (BM321) as determined by CD203cexpression on basophils of allergic patients. PBMCs from grass pollenallergic patients were incubated with serial dilutions of Phl p 1 (lightgrey bars) or BM321 (dark grey bars). Induction of CD203c was measuredas mean florescense intensities, and calculated stimulation indices areshown on the y-axis.

FIG. 6 shows the low allergenic activity of grass pollen allergenderived fusion protein HBV_Phlp6_(—)4xP1 (BM326) as determined by CD203cexpression on basophils of allergic patients. PBMCs from grass pollenallergic patients were incubated with serial dilutions of Phl p 6 (lightgrey bars) or BM326 (dark grey bars). Induction of CD203c was measuredas mean florescence intensities, and calculated stimulation indices areshown on the y-axis.

FIGS. 7 A-7 D show Timothy grass pollen allergen-specific IgG1 responsesin mice. Groups of 4 mice were immunized with 20 ug of fusion proteins(single fusion proteins and combination of 4 fusion proteins) and 10 μgeach (Phl p1 and 5) or 5 μg each (Phl p2 and 6) of wild-type allergen atstudy week 0 and 3 followed by a boost immunization at study week 17.Antigens were administered subcutaneously in the back region of theanimals. Blood was collected at study week 0, 3, 6, 9, 12, 17, 20 and 22from the tail vein of the mice. In study weeks with immunizations bloodwas collected one day before the immunization. Immune sera of mice wereinvestigated for the presence of allergen-specific IgG1 by ELISA.Pre-Immune sera before the first immunization were negative in allanimals. Individual fusion proteins were compared to the application ofa mixture of fusion proteins.

-   -   a) FIG. 7 A shows the immune response against rPhl p 1 antigen        for HBV_Phlp1_(—)4xP5 (BM321 as single component), BM321 in a        mixture with BM322, BM325 and BM326, and rPhl p 1 immunized        mice.    -   b) FIG. 7 B shows the immune response against rPhl p 2 antigen        for HBV_Phlp2_(—)4xP3 (BM321 as single component), BM322 in a        mixture with BM321, BM325 and BM326, and rPhl p 2 immunized        mice.    -   c) FIG. 7 C shows the immune response against rPhl p 5 antigen        for HBV_Phlp5_V2 (BM325 as single component), BM325 in a mixture        with BM321, BM322 and BM326, and rPhl p 5 immunized mice.    -   d) FIG. 7 D shows the immune response against rPhl p 6 antigen        for HBV_Phlp6_(—)4xP1 (BM326 as single component), BM326 in a        mixture with BM321, BM322 and BM325, and rPhl p 6 immunized        mice.

FIGS. 8 A and 8B show the molecular and immunological characterizationof recombinant fusion proteins.

FIG. 8 A. Coomassie-stained SDS-PAGE showing four PreS fusion proteinswith Bet v1 derived peptides (lane 1: 2xPA-PreS, lane 2: 2xPB-PreS, lane3: 4xPA-PreS, lane 4: 2xPA2xPB-PreS) and the carrier PreS (lane 5).

FIG. 8 B. Nitrocellulose dotted recombinant fusion proteins and PreS areprobed with a rabbit anti-PreS serum (lane 1), rabbit preimmune-serum(lane 3) buffer control for rabbit antibodies (lane 3) and monoclonalantibodies directed against Bet v 1-derived peptide P2′ (mAb2) (lane 4)and P4′ (mAb12) (lane 5) and buffer control for monoclonal mouseantibodies (lane 6).

FIG. 9 A shows IgE reactivity of rBet v 1 and recombinant fusionproteins of PreS with Bet v 1 derived peptides. Sera from birch pollenallergic patients, from non-allergic controls and only buffer weretested for their reactivity to dot-blotted rBet v 1, the fourrecombinant fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PA2PB-PreS)and PreS alone. Bound human IgE was detected with 125I-labeledanti-human IgE antibodies. Counts per minute (cpm) corresponding tobound IgE are measured with a γ-counter and indicated at Y-axis. Boxplots show the results of 50 birch pollen allergic patients.

FIG. 9 B shows the basophil activation by rBet v1 and the four PreSfusion proteins as measured by CD 203c upregulation. Blood samples ofbirch pollen allergic patients were exposed to increasing concentrations(0.001-1 μg/ml) of antigens, anti-IgE of buffer control (Co). Results ofone representative patient are shown. CD 203c expression was determinedby FACS analysis and is displayed as stimulation index (SI (y-axis).Means of triplicate measurements are shown and standard deviations areindicated.

FIGS. 10 A-10 C show lymphoproliferative responses and cytikineproduction of PBMC of birch pollen allergic patients. PBMCs of birchpollen allergic patients have been stimulated with equimolar amounts ofrBet v 1, the Bet v 1 derived peptides PA and PB, PreS alone, and PreSfusion proteins (i.e. 2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS).Stimulation indices (SI) (y-axes) are displayed.

(A) In FIG. 10 A, SI for the highest concentration (5 μg/well of Bet v 1and equimolar amounts of the peptides, PreS and PreS fusion proteins) of6 birch pollen allergic patients are shown as box blots, where 50% ofthe values are within the boxes and non-outliers are between the bars.The lines within the boxes indicate the median values.(B) In FIG. 10 B, SI for four concentrations (1=5 μg/well, 2=2.5μg/well, 3=1.25 μg/ml, 4=0.63 μg/well of rBet v1 and equimolar amountsof the peptides, PreS and PreS fusion proteins) are shown for onerepresentative patient.(C) In FIG. 10 C, Cytokine production in supernatants of PBMCs of 6birch pollen allergic patients, stimulated with with 2.5 μg/mL of rBet v1 and equimolar amounts of peptides PA and PB, PreS and four PreS fusionproteins, have been measured. Observed concentrations (pg/mL) (y-axes)after stimulation with antigens are shown in box blots, where 50% of thevalues are within the boxes and non-outliers are between the bars. Thelines within the boxes indicate the median values.

FIGS. 11 A and 11B show the induction of IgG antibodies specific forrBet v 1 and Bet v 1 homologous allergens after subcutaneousimmunization by PreS fusion proteins in rabbits.

(A) In FIG. 11 A, rabbits have been immunized withAlumhydroxide-adsorbed (Alum) (top) or complete Freund's adjuvant(CFA)-adsorbed (bottom) fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS,2PAPB-PreS) and rBet v 1. Rabbit IgG specific for rBet v 1 has beenmeasured and mean optical density (OD) values for duplicate measurementsare displayed (y-axes) for different dilutions of rabbit anti-sera(x-axes).(B1) FIG. 11 B shows a multiple sequence alignment of Bet v 1 and Bet v1-homologous allergens in alder (Aln g 1), hazel (Cor a 1) and apple(Mal d 1). Same amino acids are indicated as dots, gaps are indicated asdashes. Percentage identity of Bet v 1 homologous allergens to Bet v 1is shown at the right side. Bet v 1-derived peptide A (PA, dashed line)and peptide B (PB, full line) are framed.(B2) In FIG. 11 C, IgG antibodies of anti-rabbit sera (rab α-2PA-PreS,rab α-2PB-PreS, rab α-4PA-PreS, rab α-2PAPB-PreS) directed against rBetv 1, rAln g 1, rCor a 1 and rMal d 1 (x-axis) have been measured byELISA. Means of duplicate measurements are shown. Optical density (OD)corresponding to allergen-specific IgG in rabbit sera (post) isdisplayed in comparison with corresponding preimmune sera (pre)(y-axes).(C) In FIG. 11 D, IgG antibodies of rabbit immunized with rBet v 1 andrecombinant fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS)directed against six Bet v 1-derived peptides (P1′-P6′) (x-axis) havebeen measured by ELISA. Means of optical densitiy (OD) values forduplicate measurements (y-axis) are displayed.

FIG. 12 shows the inhibition of Anti-2xPA2xPB-PreS rabbit serum againstallergic patients' IgE compared to rabbit serum against complete rBetv 1. The percentage inhibition of IgE binding to rBet v 1 (y-axes)obtained with anti-2xPA2xPB-PreS and anti-rBet v 1 rabbit sera weredetermined by means of inhibition ELISA and are displayed as box blots,where 50% of the values are within the boxes and nonoutliers are betweenthe bars. The lines within the boxes indicate the median values. Resultsof 21 birch pollen allergic patients are shown.

FIG. 13 shows a titration of rabbit IgG raised after immunisation withPreS-fusion proteins containing either 2 or 4 copies of a Phl p 6derived peptide. For the immunogenicity testing rabbits (New ZealandWhite rabbits) were immunized with the different fusion proteins usingaluminium hydroxide as adjuvant. The induction of specific antibodieswas monitored in ELISA assays. Results show that the fusion proteinscontaining 4 peptides are more immunogenic than the fusionproteinscontaining 2 peptides.

FIGS. 14 A-14 D show the induction of a robust IgG response directed tothe grass pollen allergens Phl p 1 (FIG. 14 A), Phl p2 (FIG. 14 B), Phlp 5 (FIG. 14 C), and Phl p 6 (FIG. 14 D) following in human grass pollenallergics following subcutaneous immunization with a vaccine formulation(BM32) comprising a mixture of the 4 hypoallergenic fusion proteins withSEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. Thedetermination of IgG was carried out by ELISA. IgG levels beforetreatment (pre) are compared to IgG levels post-treatment (post).

FIG. 15 shows the results of T-cell proliferation assays performed onT-cells from grass pollen allergic individuals after immunization with avaccine formulation consisting of a mixture of the 4 hypoallergenicfusion proteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, andSEQ ID NO. 17. The T-cell reactivity is strongly reduced or absent ifcompared to grass pollen. The y-axis of the graph reflects thestimulation index.

FIGS. 16 A and 16 B show that IgG induced by therapy with a vaccineformulation (BM32) comprising a mixture of the 4 hypoallergenic fusionproteins with SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ IDNO. 17 reduces lymphoproliferative responses to grass pollen allergensin human PBMCs.

FIG. 16 A shows the experimental set-up.

FIG. 16 B shows results from T-cell proliferation assays performed inthe absence (+serum before) and presence (+serum after) oftreatment-induced IgG. The y-axis of the graph reflects the stimulationindex. P1-P5 indicate results from different study participants.

FIG. 17 shows the set-up of a clinical study carried out in 69 grasspollen allergic individuals using the vaccine formulation BM32comprising a mixture of the 4 hypoallergenic fusion proteins with SEQ IDNo. 14, SEQ ID No. 15, SEQ ID No. 16, and SEQ ID NO. 17

FIG. 18 A shows the primary sequence of fusion protein HBV Derp2-2xP2-2xP4 (sequence ID Nr. 149)

FIG. 18 B shows the primary sequence of fusion protein HBV Derp2-3xP2-3xP4 (sequence ID Nr. 150)

FIG. 18 C shows the primary sequence of fusion protein HBV Derp23-2xP4-2xP5 (sequence ID Nr. 151)

FIG. 18 D shows the primary sequence of fusion protein HBV Der p23-4xP6(sequence ID Nr. 152)

FIG. 19 A shows the change in nasal symptoms induced by treatment with 3subcutaneous injections of the vaccine formulation BM32 comprising amixture of the 4 hypoallergenic fusion proteins with SEQ ID No. 14, SEQID No. 15, SEQ ID No. 16, and SEQ ID NO. 17. Black bars: beforetreatment, grey bars: after treatment.

FIG. 19 B shows the change in the mean wheal area between titrated skinprick test before and after treatment with the vaccine formulation BM32.The titrated skin prick test was carried out using 8 serial dilutions ofgrass pollen extract (undiluted to 1:128).

FIG. 20 shows IgE binding of the Der p 2 derived peptides in comparisonto the complete allergen tested by an IgE dot-blot assay. Sera from 26house dust mite allergic patients were incubated with dottedKLH-conjugated peptides and bound IgE was detected with 125I-labelledanti-human IgE. No IgE binding was detected for any of the 5 peptides asin example 26.

EXAMPLES Example 1 Construction of Expression Plasmid forHBV_Phlp1_(—)4xP5 (BM321)

The synthetic BM321 gene were assembled from synthetic oligo-nucleotidesand/or PCR products and was cloned into an appropriate standard vector(pMK-RQkanR). The plasmid was purified from a transformed E. coli K12strain (DH10B-T1R) and concentration was determined by UV spectroscopy.The final synthetic and codon-optimized BM321 DNA-sequence was furthercloned into the expression vector pET28b(+) using appropriaterestriction sites (NcoI site at the 5′-end and EcoRI at the 3′-end). Theplasmid DNA was purified from transformed E. coli K12 DH10B (dam+dcm+)and concentration determined by UV spectroscopy. The final construct wasverified by sequencing of the insert. A summary of plasmid data and aplasmid map of final expression vector “pBM-321” is shown below.

Summary of BM321 sequence cloned into final expression vectorpET-28b(+).

Alias name Gene Plasmid Plasmid Restriction Sequence sequence size sizename sites BM321 HBV_Phlp1_4xP5 882 bp 6153 bp pBM- NcoI/ 321 EcoRI

Example 2 Transformation of Expression Plasmid into Expression Host forHBV_Phlp1_(—)4xP5 (BM321)

Chemically competent E. coli BL21(DE3) cells were transformed with theexpression plasmid by heat shock method. Transformed cells were platedon LB-agar-plates consisting of 0.5% sodium chloride 1% soy peptone,0.5% yeast extract, 1.5% agar and 50 μg/mL kanamycin for selection.Cells on LB plates were grown by over-night cultivation at 37° C. Singlecolonies of transformed BL21(DE3) E. coli cells were isolated, culturedin LB-medium and screened for growth and expression of BM321. The bestperforming clone was selected for the further establishment of a MasterCell Bank.

Example 3 Preparation of a Master Cell Bank for HBV_Phlp1_(—)4xP5(BM321)

An aliquot of the selected clone was used for inoculation of 150 mLculture medium (composition: 0.5% sodium chloride, 1% soy peptone, 0.5%yeast extract, 50 μg/mL kanamycin). The Master Cell Bank (MCB) culturewas incubated at 37° C. under constant agitation at 200 rpm until theculture reached an optical density of OD₆₀₀=1-2. Glycerol was added inorder to obtain a final glycerol concentration of 15% v/v and the MCBwas aliquoted into 1 mL vials and stored in an ultra deep freezer at−75±10° C.

Example 4 High Cell Density Fed-Batch Fermentation of HBV_PhlP1_(—)4xP5(BM321)

Synthetic culture medium (100 mL, pH=6.8, salts and trace elements, 10g/L glucose as carbon source) was inoculated with 1 mL of Master CellBank (E. coli BL21(DE3)/pBM321) and cultured in a shake flask (37° C.,200 rpm) until an optical density target value of OD=1 was reached. A 22L stainless steel fermenter was used to perform the fed-batchfermentation. For automatic and reproducible feed control, a recipe wasprogrammed allowing to pre-define specific growth-rate, feed rate,duration of batch-phase and duration of exponential feed-phase. In orderto increase the oxygen transfer rate of the fermenter, back-pressure wascontrolled and set to 1 bar. The fermenter was in-situ sterilized withthe synthetic culture medium as mentioned above and the fermentation wasstarted by inoculation with preculture. After depletion of glucose, theexponential feeding phase was started in order to maintain a specificgrowth rate of μ=0.25 h⁻¹. At an OD=45, the expression of recombinantBM321 was induced by the bolus addition of IPTG (0.8 mM finalconcentration). The culture was harvested at OD₆₀₀=73. BM321 producttiter obtained from the fed-batch fermentation was 1.2 g per L culturebroth. Afterwards, the bacterial culture broth was cooled down to ≦20°C. and centrifuged at 7,000 rpm (5,500 g) at 4° C. for 15 min. Wet cellbiomass was aliquoted and stored at −75° C.

Example 5 Cell Disruption and Clarification

For cell disruption, 748 gram biomass from Example 6 were thawed andsubdivided into aliquots a 125 gram and resuspended in a homogenizationbuffer (20 mM Tris, 1 mM EDTA, 0.1% Triton X-100, pH 11.0) undermechanical agitation at room temperature for 30 min. For celldisruption, a freeze/thaw procedure was applied by freezing −75° C. andsubsequent thawing, followed by mechanical homogenisation. The pH of thehomogenate was adjusted to pH=10.0. The crude cell homogenate wassubjected to a centrifugation step at 7,000 rpm (5,500 g) at 4° C. for30 min. The supernatants were subjected to precipitation with PEI(polyethyleneimine) under mechanical agitation. Insoluble matters wereseparated by a subsequent centrifugation step. The clarifiedsupernatants were subjected to the following chromatography step.

Example 6 Chromatographic Purification of HBV_Phlp1_(—)4xP5 (BM321)

A total of 1840 mL of the PEI precipitation supernatant from theclarification step as described in Example 7 were loaded on a 5×30 cmQ-Sepharose FF column and equilibrated with buffer A (TrisHCl, EDTA).Unbound material was removed by washing with buffer A, followed by awash with buffer C (1 sodium phosphate, EDTA, pH 7.0). Elution of theproduct fraction was accomplished by a linear gradient elution with0-100% BM32 buffer E (sodium phosphate, EDTA, NaCl pH 7.0) in BM32buffer C. Selection of product-containing fractions for pooling wasperformed according to SDS-PAGE analysis, by densitometric evaluation offraction purity and by product band intensity.

The pooled fractions from the capture step were adjusted to aconductivity of 115 mS/cm by the addition of 2.5 M sodium chloride, andthis feedstock was loaded on a Phenyl Sepharose HP column equilibratedwith buffer D (sodium phosphate, EDTA, NaCl pH 7.0). Unbound materialwas removed by washing with buffer D. Elution of the product fractionwas accomplished by a gradient elution from 40-100% buffer C (sodiumphosphate, EDTA, pH 7.0) in buffer D. Selection of product-containingfractions for pooling was performed according to SDS-PAGE analysis, bydensitometric evaluation of fraction purity and by product bandintensity.The pooled fractions from the intermediate step were adjusted to aconductivity of 80 mS/cm by the addition of 2.5 M sodium chloride, andthis feedstock was loaded on a Toyopearl Butyl 650-S column equilibratedwith a mixture buffer F (sodium phosphate, EDTA, NaCl pH 7.0). Unboundmaterial was removed by a gradient wash with 80-0% BM32 buffer F inbuffer C (sodium phosphate, EDTA, pH 7.0). Elution of the fraction wasaccomplished by a gradient elution from 0-1 buffer G (sodium phosphate,EDTA, isopropanol, pH 7.0) in buffer C. Selection of product-containingfractions for pooling was performed according to SDS-PAGE analysis, bydensitometric evaluation of fraction purity and by product bandintensity.

Example 7 Manufacturing of HBV_Phlp2_(—)4xP3 (BM322), HBV_Phlp5_V2(BM325), and HBV_Phlp6_(—)4xP1 (BM326)

For expression and manufacturing of the recombinant molecules accordingto the invention, namely HBV_Phlp2_(—)4xP3 (BM322), HBV_Phlp5_V2(BM325), and HBV_Phlp6_(—)4xP1 (BM326), the same, similar or comparablemethods and procedures as described in Example 1, Example 2, Example 3,Example 4, Example 5 and Example 6 were applied.

Example 8 Preparation of an Injectable Formulation Consisting of aMixture of HBV_PhlP1_(—)4xP5 (BM321); HBV_PhlP2_(—)4xP3 (BM322),HBV_PhlP5_V2 (BM325), and HBV_PhlP6_(—)4xP1 (BM326)

Each of the recombinant purified proteins was dissolved in an isotonicbuffer containing 0.9% sodium chloride and 2 mM sodium phosphate and toeach protein solution an appropriate amount of aluminium hydroxide wasadded. A mixture containing equal parts of the four resultingsuspensions was prepared and aliquoted under sterile condition intosealed vials. The injectable formulation obtained by this procedurecontained 0.4 mg/mL of each HBV_Ph1P1_(—)4xP5; HBV_Ph1P2_(—)4xP3,HBV_Ph1P5_V2 and HBV_Ph1P6_(—)4xP1.

Example 9 Preparation of his-Tagged HBV_Betv1_(—)4xPA

The gene coding for fusion proteins consisting of PreS fused with Bet v1-derived peptide PA twice at the N- and C-terminus (i.e. 4PA-PreS) wassynthesized by ATG:biosynthetics, Merzhausen, Germany and inserted intothe NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). The DNAsequences were confirmed by means of automated sequencing of both DNAstrands (Microsynth, Balgach, Switzerland).

The fusion protein was expressed in E coli strain BL21 (DE3; Stratagene,La Jolla, Calif.). Cells were grown in Luria Bertani-medium containing50 μg/mL kanamycin to an OD of 0.6. Protein expression was induced byadding isopropyl-β-D-thiogalactopyranoside to a final concentration of 1mmol/L over night at 37° C. Cells were harvested by centrifugation at3500 rpm for 10 minutes. The protein product was mainly detected in theinclusion body fraction. It was solubilized in 6M GuHCl, 100 mM NaH2PO4,10 mM TRIS, pH 8.0 over night. The homogenate was centrifuged at 14,000g for 18 minutes. Supernatants of were incubated with 2 mL of apreviously equilibrated Ni-NTA resin for 4 hours (Qiagen, Hilden,Germany) and the suspensions were subsequently loaded onto a column,washed with 2 column volumes of washing buffer (8 mol/L urea, 100 mmol/LNaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), and eluted with the samebuffer (pH=3.5). The purified protein was dialyzed against water.

The purity of recombinant proteins was analyzed by Coomassie-stainedSDS-PAGE (12.5%) under reducing conditions.

The identity of the fusion protein was confirmed by the means of dotblot using monoclonal antibodies, specific for Bet v 1-derived peptidesP2′ (mAb2) and P4′ (mAb12) and PreS-specific rabbit antibodies as wellas corresponding rabbit preimmune IgGs. One μg of PreS fusion proteins,PreS and HSA (control) have been immobilized on nitrocellulose and wereincubated with monocolonal as well as rabbit sera diluted 1:1000 have at4° C. Bound antibodies were detected with iodine ¹²⁵-labelled rabbitanti-mouse IgG (mAb2, mAb12) or ¹²⁵I-goat anti-rabbit IgG (rabbitanti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted1:500 for 2 hours and visualized by autoradiography. Furthermore ELISAplates (Maxisorp, Nunc, Denmark) were coated with 2 μg of PreS fusionprotein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6 washedwith PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times and blockedfor 2 hours with 1% BSA-PBST. Subsequently plates were incubated withmAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1 antibodiesin a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA in PBST)overnight at 4° C. After washing 5 times, bound IgG antibodies have beendetected with a HRP-labelled sheep anti-mouse antibody (for mAb2, mAb12)or HRP-labelled donkey anti-rabbit antibody (rabbit sera) (both GEHealthcare, Uppsala, Sweden) and colour reaction was developed.

Example 10 Preparation of his-Tagged HBV_Betv1_(—)2 xPA2xPB (BM31)

Genes coding for fusion protein consisting of PreS fused twice with Betv 1-derived peptides at the N- and C-terminus 2xPA2xPB-PreS) wassynthesized by GenScript Piscataway, N.J., USA, 2PAPB-Pres) and insertedinto the NdeI/XhoI sites of the vector pET-17b (Novagen, Germany). TheDNA sequences were confirmed by means of automated sequencing of bothDNA strands (Microsynth, Switzerland).

The recombinant PreS fusion proteins was expressed in E coli strain BL21(DE3; Stratagene, Calif.). Cells were grown in Luria Bertani-mediumcontaining 50 μg/mL kanamycin to an OD of 0.6. Protein expression wasinduced by adding isopropyl-β-D-thiogalactopyranoside to a finalconcentration of 1 mmol/L over night at 37° C. Cells were harvested bycentrifugation at 3500 rpm for 10 minutes. Proteins were mainly detectedin the inclusion body fraction. The resulting protein was solubilized in6M GuHCl, 100 mM NaH2PO4, 10 mM TRIS, pH 8.0 over night. The homogenatewas centrifuged at 14,000 g for 18 minutes. Supernatants of wereincubated with 2 mL of a previously equilibrated Ni-NTA resin for 4hours (Qiagen, Hilden, Germany) and the suspensions were subsequentlyloaded onto a column, washed with 2 column volumes of washing buffer (8mol/L urea, 100 mmol/L NaH2PO4, and 10 mmol/LTris-HCl [pH=6.1]), andeluted with the same buffer (pH=3.5). Protein was dialyzed against 10 mMNaH2PO4.

The purity of recombinant proteins was analyzed by Coomassie-stainedSDS-PAGE (12.5%) under reducing conditions. The identity of the fusionproteins was confirmed by the means of dot blot using monoclonalantibodies, specific for Bet v 1-derived peptides P2′ (mAb2) and P4′(mAb12) and PreS-specific rabbit antibodies as well as correspondingrabbit preimmune IgGs. One μg of PreS fusion protein, PreS and HSA(control) have been immobilized on nitrocellulose and were incubatedwith monocolonal as well as rabbit sera diluted 1:1000 have at 4° C.Bound antibodies were detected with iodine 125-labelled rabbitanti-mouse IgG (mAb2, mAb12) or 125I-goat anti-rabbit IgG (rabbitanti-PreS, rabbit preimmune) (Perkin-Elmer, Waltham, Mass.) diluted1:500 for 2 hours and visualized by autoradiography. Furthermore ELISAplates (Maxisorp, Nunc, Rosklide, Denmark) were coated with 2 μg of PreSfusion protein and PreS, diluted in 0.1 mol/L carbonate buffer, pH 9.6washed with PBS containing 0.05% vol/vol Tween 20 (PBST) 3 times andblocked for 2 hours with 1% BSA-PBST. Subsequently plates were incubatedwith mAb2, mAb12, anti-PreS rabbit serum and rabbit anti-Bet v 1antibodies in a dilution of 1:5000 (dilution buffer: 0.5% wt/vol BSA inPBST) overnight at 4° C. After washing 5 times, bound IgG antibodieshave been detected with a HRP-labelled sheep anti-mouse antibody (formAb2, mAb12) or HRP-labelled donkey anti-rabbit antibody (rabbit sera)(both GE Healthcare, Uppsala, Sweden) and colour reaction was developed.

Example 11 Detection of IgE Reactivity of Fusion ProteinHBV_Phlp1_(—)4xP5 (BM3212

IgE binding in comparison to the complete allergen was tested by IgEdot-blot assay. Sera from grass pollen allergic patients were incubatedwith dotted proteins and bound IgE was detected with 125I-labelledanti-human IgE. No IgE binding was detected for HBV_Phlp1_(—)4xP5(BM321) as shown in FIG. 4A.

Example 12 Detection of IgE Reactivity of Fusion ProteinHBV_Phlp2_(—)4xP3 (BM322)

IgE binding in comparison to the complete allergen was tested by IgEdot-blot assay. Sera from grass pollen allergic patients were incubatedwith dotted proteins and bound IgE was detected with 125I-labelledanti-human IgE. No IgE binding was detected for HBV_Phlp2_(—)4xP3(BM321) as shown in FIG. 4B.

Example 13 Detection of IgE Reactivity of Fusion Protein HBV_Phlp5_V2(BM3252

IgE binding in comparison to the complete allergen was tested by IgEdot-blot assay. Sera from grass pollen allergic patients were incubatedwith dotted proteins and bound IgE was detected with 125I-labelledanti-human IgE. No IgE binding was detected for HBV_(—) Phlp5_V2 (BM325)as shown in FIG. 4C.

Example 14 Detection of IgE Reactivity of Fusion ProteinHBV_Phlp6_(—)4xP1 (BM326)

IgE binding in comparison to the complete allergen was tested by IgEdot-blot assay. Sera from grass pollen allergic patients were incubatedwith dotted proteins and bound IgE was detected with 125I-labelledanti-human IgE. No IgE binding was detected for HBV_Phlp1_(—)4xP1(BM326) as shown in FIG. 4D.

Example 15 Detection of IgE Reactivity of Fusion ProteinHBV_etV1_(—)4xPA und HBV_Betv1_(—)2 xPA2xPB (BM31)

IgE binding in comparison to the complete allergen was tested by IgEdot-blot assay. Sera from grass pollen allergic patients were incubatedwith dotted proteins and bound IgE was detected with 125I-labelledanti-human IgE. No IgE binding was detected for both fusion proteins asshown in FIG. 5

Example 16 Rabbit Anti-r89P5 Antibodies Block Patient's IgE-Binding torPhl p 1

To determine the ability of peptide-induced rabbit Ig to inhibit thebinding of allergic patients' IgE antibodies to rPhl p 1, ELISA plateswere coated with 1 μg/ml rPhl p 1, washed and blocked. The plates werepreincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp1_(—)4xP5,KLHP5), a rabbit anti rPhl p 1 and, for control purposes, with thecorresponding preimmune sera. After washing, plates were incubated withhuman sera from Phl p 1-allergic patients (1:3 diluted) and bound IgEwas detected with mouse anti-human IgE (Pharmingen 1:1000) and then withsheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. Thepercentage of inhibition of IgE-binding achieved by preincubation withthe anti-peptide antisera was calculated as follows:100-OD_(i)/OD_(P)×100.

OD_(i) and OD_(P) represent the extinctions after preincubation with therabbit immune and preimmune serum, respectively. Table 1 shows thecapacity of anti-Phl p 1 peptide antibodies to inhibit the binding of 13allergic patients' IgE to complete rPhl p 1. Anti-fusion protein serablocked the IgE-binding to the same extent as sera against rPhl p 1 andKLHP5. Table 2 shows the inhibition (in %) of all 13 patients.

TABLE 1 % inhibition of 13 patients' IgE-binding to rPhl p 1 afterincubation with rabbit anti-rPhl p 1, anti-HBV_Phlp1_4xP5 and anti-KLHP5antisera % inhibition patient rPhl p 1 HBV_Phlp1_4xP5 KLHP5 1 83.6386.11 85.17 2 88.74 95.69 93.85 3 95.66 96.80 98.42 4 97.43 97.72 96.295 92.77 90.84 88.45 6 93.56 91.93 90.07 7 95.00 94.56 96.84 8 85.2589.10 90.05 9 97.07 104.72 93.73 10  91.55 103.02 95.47 11  98.85 102.43100.49 12  94.01 92.12 93.91 13  87.75 59.62 42.98 Mean 92.41 92.5989.67

Example 17 Rabbit Anti-HBV_Phlp2_(—)4xP3 Antibodies Block Patient'sIgE-Binding to rPhl p 2

To determine the ability of peptide-induced rabbit Ig to inhibit thebinding of allergic patients' IgE antibodies to rPhl p 2, ELISA plateswere coated with 1 μg/ml rPhl p 2, washed and blocked. The plates werepreincubated with 1:100-diluted rabbit anti-peptide (HBV_Phlp2_(—)4xP3,KLHP3), a rabbit anti rPhl p 2 and, for control purposes, with thecorresponding preimmune sera. After washing, plates were incubated withhuman sera from Phl p 2-allergic patients (1:3 diluted) and bound IgEwas detected with mouse anti-human IgE (Pharmingen 1:1000) and then withsheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. Thepercentage of inhibition of IgE-binding achieved by preincubation withthe anti-peptide antisera was calculated as follows:100−OD_(i)/OD_(P)×100.

OD_(i) and OD_(P) represent the extinctions after preincubation with therabbit immune and preimmune serum, respectively. Table 2 shows thecapacity of anti-Phl p 2 peptide antibodies to inhibit the binding of 19allergic patients' IgE to complete rPhl p 2. Anti-fusion protein serablocked the IgE-binding to the same extent as sera against rPhl p 2 andKLHP3. Table 2 shows the inhibition (in %) of all 19 patients.

TABLE 2 % inhibition of 19 patients' IgE-binding to rPhl p 2 afterincubation with rabbit anti-rPhl p 1, anti-HBV_Phlp2_4xP3 and anti-KLHP3antisera % inhibition patient rPhl p 2 HBV_Phlp2_4xP3 KLHP3 1 98.2481.36 2 97.50 83.90 3 96.46 98.57 90.58 4 98.31 86.77 5 96.46 81.17 699.43 72.45 9 91.25 91.38 90.44 8 95.78 54.49 9 98.60 87.55 10  95.4582.68 11  91.36 96.70 78.21 12  98.47 90.21 13  97.67 93.20 14  96.5785.64 15  97.00 91.35 16  93.73 98.06 83.62 17  95.55 76.27 18  95.9186.49 19  95.90 83.99 Mean 93.20 97.19 83.18

Example 18 Rabbit Anti-HBV_Phlp5_V2 Antibodies Block Patient'sIgE-Binding to rPhl p 5

To determine the ability of peptide-induced rabbit Ig to inhibit thebinding of allergic patients' IgE antibodies to rPhl p 5, ELISA plateswere coated with 1 μg/ml rPhl p 5, washed and blocked. The plates werepreincubated with 1:100-diluted rabbit anti-peptide (HBV_Phl p2_V2), arabbit anti rPhl p 5 and, for control purposes, with the correspondingpreimmune sera. After washing, plates were incubated with human serafrom Phl p 5-allergic patients (1:3 diluted) and bound IgE was detectedwith mouse anti-human IgE (Pharmingen 1:1000) and then with sheepanti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentageof inhibition of IgE-binding achieved by preincubation with theanti-peptide antisera was calculated as follows: 100−OD_(i)/OD_(P)×100.

OD_(i) and OD_(P) represent the extinctions after preincubation with therabbit immune and preimmune serum, respectively. Table 3 shows thecapacity of anti-Phl p 5 peptide antibodies to inhibit the binding of 16allergic patients' IgE to complete rPhl p 5. Anti-fusion protein serablocked the IgE-binding to the same extent as sera against rPhl p 5 andbetter than KLH peptide mix. Table 3 shows the inhibition (in %) of all16 patients.

TABLE 3 % inhibition of 13 patients' IgE-binding to rPhl p 5 afterincubation with rabbit anti-rPhl p 1, anti-HBV_Phlp5_V2 and anti-KLHpeptide mix antisera % inhibition patient rPhl p 5 HBV_Phlp5_V2 KLHPmix1 99.00 96.69 91.74 2 94.57 94.15 68.42 3 98.98 95.88 85.74 4 97.3988.38 80.23 5 98.95 93.74 62.33 6 98.52 93.36 78.82 9 97.22 91.35 79.948 96.02 89.70 80.14 9 97.09 88.48 61.11 10  99.30 84.03 92.92 11  99.5094.09 86.46 12  95.45 88.97 81.31 13  96.22 93.34 60.87 14  90.86 94.8083.02 15  98.45 94.15 83.60 16  94.68 92.46 91.77 Mean 97.01 92.10 79.28

Example 19 Rabbit Anti-HBV_Phlp6_(—)4xP1 Antibodies Block Patient'sIgE-Binding to rPhl p 6

To determine the ability of peptide-induced rabbit Ig to inhibit thebinding of allergic patients' IgE antibodies to rPhl p 6, ELISA plateswere coated with 1 μg/ml rPhl p 6, washed and blocked. The plates werepreincubated with diluted rabbit anti-peptide (HBV_Phlp6_(—)4xP1,KLHP1), a rabbit anti rPhl p 6 and, for control purposes, with thecorresponding preimmune sera. After washing, plates were incubated withhuman sera from Phl p 6-allergic patients (1:3 diluted) and bound IgEwas detected with mouse anti-human IgE (Pharmingen 1:1000) and then withsheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. Thepercentage of inhibition of IgE-binding achieved by preincubation withthe anti-peptide antisera was calculated as follows:100−OD_(i)/OD_(P)×100. OD_(i) and OD_(P) represent the extinctions afterpreincubation with the rabbit immune and preimmune serum, respectively.Table 4 shows the capacity of anti-Phl p 6 peptide antibodies to inhibitthe binding of 21 allergic patients' IgE to complete rPhl p 6.Anti-fusion protein sera blocked the IgE-binding to the same extent assera against rPhl p 6 and KLHP1. Table 4 shows the inhibition (in %) ofall 21 patients.

TABLE 4 % inhibition of 21 patients' IgE-binding to rPhl p 6 afterincubation with rabbit anti-rPhl p 6, anti-HBV_Phlp6_4xP1 and anti-KLHP1antisera % inhibition patient rPhl p 6 HBV_Phlp6_4xP1 KLHP1  1 96.5295.96 95.64  2 88.26 91.20 88.06  3 95.07 95.39 94.10  4 82.77 83.7481.98  5 96.71 96.35 95.20  6 95.46 93.38 92.83  7 90.52 88.07 86.06  886.69 85.14 83.08  9 89.09 91.56 89.00 10 97.05 96.48 97.42 11 86.9789.19 84.95 12 37.22 49.14 44.90 13 75.97 79.19 75.85 14 91.05 92.1387.93 15 89.01 88.25 85.82 16 92.46 91.82 91.30 17 78.99 84.13 77.93 1847.25 67.02 67.825 19 93.84 86.62 79.841 20 58.42 56.69 71.388 21 39.9256.69 67.797 Mean 81.39 83.36 82.81

Example 20 IgE Reactivity of PreS Fusion Proteins Determined by Dot Blotand ELISA

Purified rBet v 1, recombinant fusion proteins 4xPA-PreS, 2xPA2xPB-PreSwere tested for their IgE reactivity by RAST-based, non-denaturing dotblot assays. Two μg of the purified proteins and, for control purposes,HSA were dotted onto nitrocellulose membrane strips (Schleicher &Schuell, Dassel, Germany).

Nitrocellulose strips were blocked in buffer A (Vrtala, J Clin Invest,1997) and incubated with sera from birch pollen allergic patients(n=50), sera from non-allergic persons (n=3) diluted 1:10, buffercontrol and positive control (1:1000 diluted rabbit anti-rBet v 1antiserum). Bound IgE antibodies were detected with ¹²⁵I-labelledanti-human IgE antibodies (BSM Diagnostica, Vienna, Austria), boundrabbit antibodies with a ¹²⁵I-labeled goat anti-rabbit antiserum(Perkin-Elmer) and visualized by autoradiography (Valenta et al., 1992).Additionally, ELISA plates were coated with rBet v 1 and the purifiedPreS fusion proteins (5 μg/mL). After washing and blocking as describedabove, plates were incubated with sera of birch pollen allergic patients(n=21) and three non-allergic control sera diluted 1:5. Bound IgE wasdetected by purified mouse anti human IgE (BD Pharmingen) diluted 1:1000overnight and visualized with HRP-labelled sheep anti mouse IgG (GEHealthcare) diluted 1:2000. After washing, colour reaction wasdetermined as described above.

Example 21 Allergen-Induced Upregulation of CD203c of Allergic Patients'Basophils

Heparinized blood samples were obtained from birch allergic patientsafter informed consent was given and were incubated with increasingconcentrations of rBet v 1, 4PA-PreS, 2PAPB-PreS ranging from 0.001 to 1mg/mL, a monoclonal anti-IgE antibody (Immunotech, Marseille, France) aspositive control, or PBS (negative control) for 15 min (37° C.). CD 203cexpression was determined as previously described.

Example 22 Lymphoproliferative Responses and Cytokine Induction in PBMCfrom Birch Pollen Allergic Patients

PBMCs from birch pollen allergic patients (n=6) have been isolated byFicoll (Amersham Biosciences, Uppsala, Sweden) density gradientcentrifugation. Subsequently PBMCs were resuspended in AIM V medium(Life Technologies, Grand Island, N.Y.) to a final concentration of2×10⁵ cells/well and stimulated with decreasing antigen doses (equimolaramounts of 5 μg/well rBet v 1, PA, PB, PreS, 2PA-PreS, 2PB-PreS,4PA-PreS, 2PAPB-PreS), with medium alone (negative control) or with IL-2(4 IE/well) (positive control). After 6 days, proliferative responseswere measured by [³H] thymidine incorporation and are expressed asstimulation indices (SI).

Furthermore cytokine production of 17 different cytokines (i.e. IL-113,IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IFN-γ,TNF-α, G-CSF, GM-CSF, MIP-1β, MCP-1) has been measured after 6 days ofstimulation with Bio-plex Pro Human Cytokine 17-Plex Panel (Bio-RadLaboratories) according the manufacturer's instructions. Briefly, theundiluted supernatants were mixed with anti-cytokine/chemokine mousemonoclonal antibodies coupled to different beads as capture antibodies(Bio-Rad). An 8-point standard curve was used to achieve low-endsensitivity. After washing, anti-cytokine biotinylated detectionantibody was added. The reaction was visualized by addingStreptavidin-labelled Phycoerythrin (PE) and assay buffer. The sampleswere analyzed on a Luminex 100 instrument (Biosource, Nivelles, Belgium)and the data were acquired using the Bio-Plex Manager 6.0 software. Allsamples were analyzed in one run. Results are shown in FIGS. 10 A-10 C.

Example 23 Analysis of Rabbit Sera Immunized with rBet v 1 and PreSFusion Proteins for their Recognition of rBet v 1, Bet v 1 HomologousAllergens and Bet v 1-Derived Peptides by ELISA

ELISA plates (Maxisorp, Nunc) were coated either with 1 μg/ml rBet v 1or homologous allergens in alder (rAln g 1), hazel (rCor a 1), apple(rMal dl) and additionally with several Bet v 1-derived peptides in aconcentration of 1 μg/ml overnight at 4° C. After washing and blockingas described above sera from rabbits immunized with rBet v 1 and thePreS fusion proteins conjugated to alum or CFA, were incubated in serial1:2 dilutions ranging from 1:500 to 1:1 280 000 and in a concentrationof 1:1000. Bound rabbit IgG was detected with HRP-labelled donkeyanti-rabbit antibodies (GE Healthcare) and colour reaction wasdetermined as described above.

Example 24 Inhibition of Allergic Patients' IgE Binding to rBet v 1

An inhibition ELISA was used to study the inhibition of the binding ofbirch pollen allergic patients' IgE to rBet v 1. ELISA plates werecoated with rBet v 1 in a concentration of 1 μg/m at 4° C. overnight.After washing and blocking plates were pre-incubated with rabbit seradirected against the PreS fusion protein 2PAPB-PreS and anti-Bet v 1rabbit serum in a dilution of 1:80 and 1:160 in comparison with rabbitpreimmune sera overnight at 4° C. After an additional washing step seraof birch pollen allergic patients diluted 1:5 were added overnight at 4°C. and bound human IgE were detected with a 1:1000 diluted alkalinephosphatase-conjugated mouse monoclonal anti human IgE antibody (BDPharmingen). The percentage of inhibition of IgE binding to rBet v 1after pre-incubation with 2PAPB-PreS rabbit antisera and Bet v 1 rabbitantisera was calculated as follows: percentinhibition=100−(OD^(i)×100/OD^(P)). OD^(P) and OD^(i) represent theextinctions after pre-incubation with specific rabbit IgG (OD^(i)) orpreimmune sera (OD^(P)), respectively. (FIG. 12)

Example 25 Use of a Vaccine Formulation Comprising a Mixture of 4Hypoallergenic Fusion Proteins for the Treatment of Grass Pollen Allergyin Grass Pollen Allergic Human Individuals

An injectable formulation of hypoallergenic fusion proteins SEQ IDNo.14, SEQ ID No. 15, SEQ ID No.16, and SEQ ID No. 17 with aluminumhydroxide was prepared as described in example 8. In the course of aclinical study, the vaccine was administered 3 times subcutaneously to69 grass pollen allergic human subjects. (FIG. 17)

Vaccination with the vaccine formulation led to a robust IgG immuneresponse. Induction of allergen-specific IgG following s.c. injection ofthe 3 different dose levels of the vaccine and placebo was determined byELISA in the sera collected from the study participants before and aftertreatment with 3 s.c. injections of the vaccine formulation. (FIGS. 14A-14 D).

For this purpose, ELISA plates (Nunc Maxisorp, Roskilde, Denmark) werecoated with 5 μg/ml of the antigens Phl p 1, Phl p 2, Phl p 5, and Phl p6 or human serum albumin (HSA) as control over night at 4° C. Afterwashing with PBS containing 0.5% Tween 20 (PT) and blocking with 2% w/vBSA in PT, plates were subsequently incubated with 1:10 to 1:100 dilutedsera from patients, serum from a non-atopic individual or buffer alonein triplicates overnight at 4° C. Bound IgE antibodies were detectedwith HRP-coupled anti-human IgE antibodies diluted in PT, 0.5% w/v BSA.The colour development was performed by addition of staining solutionABTS (2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammoniumsalt; Sigma-Aldrich, St. Louis, Mo., USA) (100 μl/well). The opticaldensity was measured using an ELISA Reader at 405 nm. The results of IgGassessments are shown in FIGS. 14 A-14 D.

The vaccine did not provoke any relevant T-cell reactivity towards thehypoallergenic fusion proteins present in the vaccine formulation asdetermined by in-vitro T-cell proliferation assay (FIG. 15), thusdemonstrating the lack of T-cell reactivity of the hypoallergenic fusionproteins.

T-cell proliferation assays were performed using the followingprocedure: Peripheral blood mononuclear cells (PBMC) were isolated fromheparinised blood samples of the grass pollen allergic patients byFicoll (Amersham Pharmacia Biotech, Little Chalfont, UK) densitygradient centrifugation. PBMC (2×10⁵) were then cultured in triplicatesin 96-well plates (Nunclone; Nalge Nunc International, Roskilde,Denmark) in 200 μl serum-free Ultra Culture medium (BioWhittaker,Rockland, Me.) supplemented with 2 mM L-glutamin (SIGMA, St. Louis,Mo.), 50 μM b-mercaptoethanol (SIGMA) and 0.1 mg gentamicin per ml(SIGMA) at 37° C. and 5% CO₂ in a humidified atmosphere. Cells werestimulated with a mixture containing 0.25 μg of each polypeptidecomponent of the vaccine and for comparison an equimolar concentrationsof grasspollen extract or for control purposes with 4 U Interleukin-2per well (Boehringer Mannheim, Germany) or medium alone. After 6 dculture 0.5 μCi per well [3H]thymidine (Amersham Pharmacia Biotech) wasadded and 16 h thereafter incorporated radioactivity was measured byliquid scintillation counting using a microbeta scintilllation counter(Wallac ADL, Freiburg, Germany). Mean cpm were calculated from thetriplicates and stimulation indices (SI) were calculated as the quotientof the cpm obtained by antigen or interleukin-2 stimulation and theunstimulated control. Results of proliferation assays are shown in FIG.15.

Treatment with the vaccine induced IgG antibodies with the capability tomodulate the allergen-specific T-cell response as demonstrated by areduced proliferative response upon stimulation with grass pollenallergens in the presence of treatment-induced IgG. (FIGS. 16 A and 16B).

For this purpose, T-cell proliferation assays were performed with PBMCsisolated from study participants after treatment as described above withthe exception that the stimulation was done with a mixture of the 4grass pollen allergens Phl p 1, Phl p 2, Phl p5, and Phl p 6 (0.25 μgper allergen) together with serum collected from the same participantbefore and after the treatment. The experimental set-up and results areshown in FIGS. 16 A and 16 B.

Reduction of nasal allergy symptoms induced by provocation in a pollenchamber and reduction of skin reactivity as determined by titrated skinprick testing was observed in patients having received 3 injectionscontaining either 20 μg or 40 μg of each of the 4 polypeptides whilethere was no reduction in those parameters after treatment with doses of10 μg of each polypeptide. (see FIGS. 19 A and 19 B).

Example 26 Selection of Peptides Derived from House Dust Mite AllergenDer p 2 and Design of PreS Fusion Proteins Using Those Peptides

The 5 non IgE binding Der p 2 derived peptides—Der p2 Pep1 (SEQ IDNo.96), Der p2 Pep2 (SEQ ID No.97), Der p2 Pep3 (SEQ ID No. 98), Der p2Pep4 (SEQ ID No. 99), and Der p2 Pep5 (SEQ ID No. 100)—were screenedwith respect to

-   -   their IgE binding properties (dot blot assay)    -   their potential to induce Der p 2 specific T-cell reactions, and        (T-cell proliferation assay)    -   their ability to induce Der p 2-specific antibodies with the        capacity to block human patient's IgE to Der p 2. (inhibition        ELISA using rabbit anti-peptide IgG)

For that purpose, each of the peptides was chemically coupled to KLH.KLH and chemical coupling of the peptides was used in this screeningexperiment because it is an easy-to-use and well established andstraight forward model system allowing initial comparison of thedifferent peptides.

IgE binding of the Der p 2 derived peptides in comparison to thecomplete allergen was tested by IgE dot-blot assay. Sera from 26 housedust mite allergic patients were incubated with dotted KLH-conjugatedpeptides and bound IgE was detected with 125I-labelled anti-human IgE.No IgE binding was detected for any of the 5 peptides as shown below.

To identify peptides which induce a low lymphoproliferative response inPBMC from house dust mite allergic patients PBMCs isolated from 10patients were stimulated with the 5 Der p 2 derived peptides alone, theKLH-conjugated peptides, and wild-type Der p 2 for comparison.

PBMCs from all 10 patient were stimulated by the wild-type Der p 2, andthere was no or only very low proliferation upon stimulation with Der p2Pep1, Der p2 Pep2, and Der p2 Pep4. Stimulation with Der p2 Pep3 and Derp2 Pep5 however, resulted in significant proliferation of the PBMCs in 4out of 10 and 3 out of 10 cases, respectively, indicating that peptides3 and 5 contain important T-cell epitopes.

To identify the ability of the peptides to induce blocking IgG, rabbitswere immunized with the 5 individual KLH-peptide conjugates.Subsequently, the ability of peptide-induced rabbit IgG to inhibit thebinding of allergic patients' IgE antibodies to rDer p 2 wasinvestigated by ELISA. ELISA plates were coated with 1 μg/ml rDer p 2,washed and blocked. The plates were preincubated with 1:100-dilutedrabbit anti-peptide (KLH-P1, KLH-P2, KLH-P3, KLH-P4, and KLH-P5), arabbit anti rDer p 2 and, for control purposes, with the correspondingpreimmune sera. After washing, plates were incubated with human serafrom house dust mite allergic, Der p 2 sensitized patients (1:3 diluted)and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000)and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience)1:2000. The percentage of inhibition of IgE-binding achieved bypreincubation with the anti-peptide antisera was calculated as follows:100−ODi/ODP×100.

TABLE 5 Inhibition capacity of anti-Der p 2-peptide antibodies toinhibit the binding of 20 allergic patients' IgE to complete rDer p 2.Anti- KLH-peptide sera induced by peptides 2, 3, and 4 blocked theIgE-binding to the same extent as sera against wild-type Der p 2. Table5 shows the inhibition (in %) of all 20 patients. Patient PeptidePeptide Peptide Peptide Peptide Der # 1 2 3 4 5 p 2 1 50.63 74.41 78.3675.50 1.07 78.26 2 49.61 77.15 82.95 77.85 4.16 82.74 3 64.73 87.4192.13 89.25 0.00 93.34 4 37.98 72.24 81.08 75.60 2.48 84.25 5 0.00 43.5650.52 47.28 0.00 56.70 6 54.12 80.63 82.64 80.94 1.10 83.21 7 51.4379.64 92.08 83.25 16.16 93.51 8 42.93 71.02 79.55 75.44 0.83 78.35 930.33 58.36 50.94 56.49 7.76 57.03 10 38.46 66.79 71.20 71.25 0.00 69.0611 48.15 74.60 83.13 78.97 5.59 83.56 12 46.06 68.54 74.05 71.32 10.0576.46 13 44.71 73.62 87.29 77.19 4.97 84.34 14 39.20 63.55 53.94 65.300.00 66.20 15 43.62 71.82 89.94 74.54 0.51 94.39 16 38.09 69.94 84.0872.45 1.29 86.83 17 43.63 74.16 87.12 78.50 2.98 89.10 18 29.09 73.7589.97 77.59 1.38 90.66 19 40.44 56.77 62.09 62.30 0.00 66.16 20 20.8960.85 70.76 63.16 2.69 74.98 mean 40.71 69.94 77.69 72.71 3.15 79.46

TABLE 6 Decision matrix for selection of peptides. Peptides 2 and 4 meetall requirements of peptide fragments of the present invention. peptideinduces IgG which peptide is peptide induces inhibit binding non-IgE noor only low of human IgE Peptide binding T-cell reactivity to Der p 2suitable? Der p2 Pep1 ✓ ✓ X no Der p2 Pep2 ✓ ✓ ✓ yes Der p2 Pep3 ✓ X ✓no Der p2 Pep4 ✓ ✓ ✓ yes Der p2 Pep5 ✓ X X no

Example 27 Selection of Der p 1 Derived Hypoallergenic Peptides

The ability of Der p 1 derived peptides to induce IgE-blocking IgGantibodies was determined using rabbit-anti-peptideKLH antisera and serafrom 6 house dust mite allergic patients in an inhibition ELISA asdescribed in example 26 with the exception that the ELISA plates werecoated with wild-type Der p 1 instead of Der p 2.

TABLE 7 Inhibition capacity of anti-Der p 1- peptide antibodies toinhibit the binding of 6 allergic patients' IgE to complete Der p 1.Anti-KLH-peptide sera induced by peptides 1, 2, and 8 were found toblock the IgE-binding to a similar extent as sera against wild-type Derp 1. Table 7 shows the inhibition (in %) of 6 patients. Patient IPatient II Patient III Patient IV Patient V Patient VI mean der p 1 72.991.3 80 90.8 87.5 89.7 85.4 peptide 1 50 68.4 65.5 87.7 77.4 85.1 72.4peptide 2 47.8 73.4 66.1 83.2 72.6 82.5 70.9 peptide 3 22.5 28.2 22.135.5 26.4 27.6 27.1 peptide 4 24.4 42.4 33.4 46.5 33.2 42 37.0 peptide 522.7 31.4 23.3 38.4 30.4 31.5 29.6 peptide 6 1.9 12.8 3.6 5.6 4.2 5.45.6 peptide 7 30 51.8 43.5 67.4 52.1 59.6 50.7 peplide 8 41.1 65.8 52.876 66.2 73.9 62.6

1-27. (canceled)
 28. A pharmaceutical formulation, comprising at leastone polypeptide comprising the amino acid sequence selected from thegroup consisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQID No.
 17. 29. The pharmaceutical formulation of claim 28, wherein theat least one polypeptide is present in an amount of 10 ng to 1 g. 30.The pharmaceutical formulation of claim 28, wherein the at least onepolypeptide is present in an amount of 100 ng to 10 mg.
 31. Thepharmaceutical formulation of claim 28, wherein the at least onepolypeptide is present in an amount of 0.5 μg to 200 μg.
 32. Thepharmaceutical formulation, further comprising at least one of anadjuvant, a pharmaceutical acceptable excipient and a preservative. 33.A polypeptide comprising the amino acid sequence selected from the groupconsisting of SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No.17.
 34. A nucleic acid molecule encoding a polypeptide of claim
 33. 35.A vector, comprising a nucleic acid molecule of claim
 34. 36. The vectorof claim 35, which is an expression vector.
 37. The vector of claim 35,which is a bacterial, fungal, insect, viral or mammalian vector.
 38. Ahost cell, comprising the nucleic acid molecule of claim
 34. 39. A hostcell, comprising the vector of claim
 35. 40. A method for treating agrass pollen allergy in a human or animal subject in need thereof, themethod comprising administering an effective amount of thepharmaceutical formulation of claim 28 to the subject.
 41. A method fortreating a grass pollen allergy in a human or animal subject in needthereof, the method comprising administering an effective amount of thepolypeptide of claim 33 to the subject.
 42. A method for treating agrass pollen allergy in a human or animal subject in need thereof, themethod comprising administering an effective amount of the nucleic acidmolecule of claim 34 to the subject.